Assays and methods of treatment relating to vitamin D insufficiency

ABSTRACT

Described herein are assays directed to determining the level of bioavailable or free vitamin D in a blood sample in a subject. The values determined for bioavailable or free vitamin D indicate whether the subject suffers from insufficient levels of vitamin D. Also described herein are methods of treatment for vitamin D insufficiency.

CROSS REFERENCE

This application is a U.S. National Phase Application under 35 U.S.C.§371 of International Patent Application No. PCT/US2012/020407, filed onJan. 6, 2012, which claims benefit of U.S. Provisional PatentApplication Ser. No. 61/430,643 filed Jan. 7, 2011. The entire contentsof the foregoing are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with federal government support under GrantNumber M01-(RR-01066)-Harvard Clinical and Translational Science Centerawarded by the National Center for Research Resources and Grant K231K23DK081677 awarded by the National Institutes of Health. The U.S.Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to assays and methods of treatment relating tovitamin D insufficiency.

BACKGROUND OF THE INVENTION

Vitamin D insufficiency (variously defined as <20 to 30 ng/mL of totalserum 25(OH)D as currently measured) is highly prevalent, even inotherwise healthy individuals. Reported in >1 billion people worldwide,it is now recognized as one of the most common subclinical medicalconditions in the world. Beyond rickets, a manifestation of severevitamin D insufficiency recognized since the 17th century,³⁵ vitamin Ddeficiency (commonly defined as <10-25 ng/mL of total serum25[OH]D)^(21,34,36) has since been associated with an increased risk ofosteoporosis, cancer, infectious disease, CVD disease, allergy, asthma,multiple sclerosis, muscle weakness, rheumatoid arthritis, and diabetes.Low vitamin D can arise from insufficient intake from nutritionalsources, insufficient synthesis (via UV-B radiation of the skin),adiposity, age, physical activity, or other disease-related factors suchas diabetes, bariatric surgery, fat malabsorption syndromes, and kidneydisease.^(14,37,38) The use of sunscreen with sun protection factor(SPF)≧30 reduces the ability of the skin to produce vitamin D by 99%,thus contributing to the pandemic.

One recent study found that vitamin D insufficiency was present in 72%of community-dwelling men older than 65 years of age, and in up to 86%of those men who were obese, lived at higher latitudes, or infrequentlyparticipated in outdoor activities.¹⁷ Although vitamin D deficiency isless common, it is estimated to affect 26%-54% of community-dwellingolder adults and 57% of hospitalized patients.^(17,36,40) A recognizedproblem in older adults, people of all ages who live in diversegeographic locations are also susceptible, including sunny climatedwellers.⁴¹ A study of younger adults with limited exposure to theoutdoors in a northeastern urban setting reported that 32% of studentsand doctors aged 18-29 years were vitamin D deficient at the end of thewinter.⁴² In diseases including diabetes, rheumatoid arthritis, renaldisease, as well as in individuals who are obese, hospitalized,pregnant, newborn, highly deficient levels of this hormone arecommon.^(40,43-45) Current recommendations for vitamin D supplementationare largely inadequate.^(17,46,47) According to Holick,⁴⁶ 25(OH)D is themost-ordered hormone assay in the US, used as the basis for treatmentrecommendations. However, assay results as well as cutoff levels of25(OH)D to define the extent of vitamin D insufficiency are subject toconsiderable variation. Given the prevalence and breadth of illnessespotentially associated with low vitamin D, gaining a betterunderstanding of vitamin D status to guide management of vitamin Dinsufficiency is a public health priority. The Institute of Medicine(IOM) has recognized that that assay variation and lack of consensusregarding cutoffs defining insufficiency/deficiency have causedconfusion about the appropriate dietary intake of vitamin D.³³ The IOMhas also cautioned against excessive intake due to the risks of kidneyand tissue damage and have urged more targeted research in this area.Importantly, the method used to determine vitamin D status should beclinically relevant and applicable across diverse populations.

SUMMARY OF INVENTION

Described herein are methods, assays, methods of treatment, and systemsrelated to determining the level of free and/or bioavailable vitamin Din a blood sample obtained from a subject.

In one aspect, the invention relates to an assay comprising a) analyzinga blood sample obtained from a subject to determine a level of VDBP(vitamin D binding protein) polypeptide, albumin polypeptide and totalvitamin D; wherein a level of bioavailable vitamin D is:=(K _(alb) *[Alb]+1)*[Free Vitamin D]and wherein a level of free vitamin D is:={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)}).

In some embodiments, a level of bioavailable vitamin D lower than 25% ofthe mean value of bioavailable vitamin D in a population of healthysubjects can indicate that the subject has a vitamin D insufficiency. Insome embodiments, the vitamin D can be selected from the groupconsisting of: 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining the level of VDBP polypeptide oralbumin polypeptide can comprise the use of a method selected from thegroup consisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.

In some embodiments, determining the level of total vitamin D cancomprise the use of a method selected from the group consisting of:radioimmunoassay; liquid chromatography tandem mass spectroscopy; enzymelinked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the invention can further comprise the step ofadministering a vitamin D insufficiency treatment to a subject who isdetermined to have a vitamin D insufficiency. In some embodiments, thetreatment can comprise administering a compound selected from the groupconsisting of: calcitriol; dihydrotachysterol; doxercalciferol;paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to an assay comprising;analyzing a blood sample obtained from a subject to determine a level ofVDBP polypeptide, albumin polypeptide and total vitamin D; wherein alevel of free vitamin D is:={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)}).

In some embodiments, a level of free vitamin D lower than 25% of themean value of free vitamin D in a population of healthy subjects canindicate that the subject has a vitamin D insufficiency. In someembodiments, the vitamin D can be selected from the group consisting of:25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining the level of VDBP polypeptide oralbumin polypeptide can comprise the use of a method selected from thegroup consisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.

In some embodiments, determining the level of total vitamin D cancomprise the use of a method selected from the group consisting of:radioimmunoassay; liquid chromatography tandem mass spectroscopy; enzymelinked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the invention can further comprise the step ofadministering a vitamin D insufficiency treatment to a subject who isdetermined to have a vitamin D insufficiency. In some embodiments, thetreatment can comprise administering a compound selected from the groupconsisting of: calcitriol; dihydrotachysterol; doxercalciferol;paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to a method for treating avitamin D insufficiency in a subject comprising detecting a level ofVDBP polypeptide, albumin polypeptide and total vitamin D in a bloodsample obtained from a subject; wherein a level of bioavailable vitaminD is:=(K _(alb) *[Alb]+1)*[Free Vitamin D]and wherein a level of free vitamin D is:={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)})and administering a treatment for vitamin D insufficiency to the subjectif the level of bioavailable vitamin D is less than 25% of the meanvalue of bioavailable vitamin D in a population of healthy subjects.

In some embodiments, the vitamin D can be selected from the groupconsisting of: 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining the level of VDBP polypeptide oralbumin polypeptide can comprise the use of a method selected from thegroup consisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.

In some embodiments, determining the level of total vitamin D cancomprise the use of a method selected from the group consisting of:radioimmunoassay; liquid chromatography tandem mass spectroscopy; enzymelinked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the treatment can comprise administering a compoundselected from the group consisting of: calcitriol; dihydrotachysterol;doxercalciferol; paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to a method for treating avitamin D insufficiency in a subject comprising detecting a level ofVDBP polypeptide, albumin polypeptide and total vitamin D in a bloodsample obtained from a subject; wherein a level of free vitamin D is:={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)})and administering a treatment for vitamin D insufficiency to the subjectif the level of free vitamin D is less than 25% of the mean value offree vitamin D in a population of healthy subjects.

In some embodiments, the vitamin D can be selected from the groupconsisting of: 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining the level of VDBP polypeptide oralbumin polypeptide can comprise the use of a method selected from thegroup consisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.

In some embodiments, determining the level of total vitamin D cancomprise the use of a method selected from the group consisting of:radioimmunoassay; liquid chromatography tandem mass spectroscopy; enzymelinked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the treatment can comprise administering a compoundselected from the group consisting of: calcitriol; dihydrotachysterol;doxercalciferol; paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to a system for obtaining datafrom at least one blood sample obtained from at least one subject, thesystem comprising: a determination module configured to receive the atleast one blood sample and perform at least one analysis on the at leastone blood sample to determine a level of bioavailable or free vitamin Din the sample; a storage device configured to store data output fromsaid determination module; and a display module for displaying a contentbased in part on the data output from said determination module, whereinthe content comprises a signal indicative of the level of bioavailableor free vitamin D.

In some embodiments, the system further comprises a means of inputting avalue for the level of one or more of VDBP polypeptide, albuminpolypeptide, and total vitamin D determined to be in a test sample. Insome embodiments, the content displayed on said display module furthercomprises a signal indicative of the subject having an increasedlikelihood of a vitamin D insufficiency if the level of bioavailable orfree vitamin D is determined to be lower than 25% of the mean value ofbioavailable vitamin D in a population of healthy subjects. In someembodiments, the content displayed on said display module furthercomprises a signal indicative of the subject being recommended toreceive a treatment for vitamin D insufficiency.

In some embodiments, a level of free and/or bioavailable vitamin D lowerthan 25% of the mean value of free and/or bioavailable vitamin D in apopulation of healthy subjects can indicate that the subject has avitamin D insufficiency. In some embodiments, the vitamin D can beselected from the group consisting of: 25-hydroxyvitamin D and1,25-dihydroxyvitamin D.

In some embodiments, determining the level of VDBP polypeptide oralbumin polypeptide can comprise the use of a method selected from thegroup consisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.

In some embodiments, determining the level of total vitamin D cancomprise the use of a method selected from the group consisting of:radioimmunoassay; liquid chromatography tandem mass spectroscopy; enzymelinked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the invention can further comprise the step ofadministering a vitamin D insufficiency treatment to a subject who isdetermined to have a vitamin D insufficiency. In some embodiments, thetreatment can comprise administering a compound selected from the groupconsisting of: calcitriol; dihydrotachysterol; doxercalciferol;paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to a method of treatmentcomprising:analyzing a blood sample obtained from a subject to determinea level of free or bioavailable vitamin D; wherein a level of free orbioavailable vitamin D lower than 25% of the mean value of free orbioavailable vitamin D in a population of healthy subjects indicatesthat the subject has a vitamin D insufficiency; and administering avitamin D insufficiency treatment to a subject who is determined to havea vitamin D insufficiency.

In some embodiments, the vitamin D can be selected from the groupconsisting of: 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining of the level of free and/or vitamin Dcan comprise the use of a method selected from the group consisting of:immunoassay; two-step immunoassay with antibody capture; one-stepimmunoassay with immobilized antibody and competitive detection;one-step immunoassay with immobilized competitior and labeled antibody;fluorescence polarization immunoassay; differential precipitation(immunoprecipitation, affinity precipitation); immunodepletion; andaffinity binding chromatography; and a method selected from the groupconsisting of: radioimmunoassay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; dye linked immunosorbent assay; liquid chromatography tandem massspectroscopy and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the treatment can comprise administering a compoundselected from the group consisting of: calcitriol; dihydrotachysterol;doxercalciferol; paricalcitol; cholecalciferol and ergocalciferol.

In another aspect, the invention relates to an assay comprisinganalyzing a blood sample obtained from a subject to determine a level offree vitamin D and albumin polypeptide; wherein a level of bioavailablevitamin D is:=(K _(alb) *[Alb]+1)*[Free Vitamin D].

In some embodiments, a level of bioavailable vitamin D lower than 25% ofthe mean value of bioavailable vitamin D in a population of healthysubjects can indicate that the subject has a vitamin D insufficiency. Insome embodiments, the vitamin D can be selected from the groupconsisting of: 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

In some embodiments, determining the level of albumin polypeptide cancomprise the use of a method selected from the group consisting of:enzyme linked immunosorbent assay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; dye linked immunosorbent assay; immunoturbidimetric assay;immunonephelometric assay; dye-based photometric assay; western blot;immunoprecipitation; radioimmunological assay (RIA); radioimmunometricassay; immunofluorescence assay and mass spectroscopy.

In some embodiments, determining of the level of free and/or vitamin Dcan comprise the use of a method selected from the group consisting of:immunoassay; two-step immunoassay with antibody capture; one-stepimmunoassay with immobilized antibody and competitive detection;one-step immunoassay with immobilized competitior and labeled antibody;fluorescence polarization immunoassay; differential precipitation(immunoprecipitation, affinity precipitation); immunodepletion; andaffinity binding chromatography; and a method selected from the groupconsisting of: radioimmunoassay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; dye linked immunosorbent assay; liquid chromatography tandem massspectroscopy and high-pressure liquid chromatography.

In some embodiments, an insufficiency of vitamin D can indicate anincreased risk of a condition selected from the group consisting of:decreased bone density; decreased bone mineral density; bone fractures;bone resorption; rickets; osteitis fibrosa cystica; fibrogenesisimperfect ossium; osteosclerosis; osteoporosis; osteomalacia; elevatedparathyroid hormone levels; parathyroid gland hyperplasia; secondaryhyperparathyroidism; hypocalcemia; infection; cancer; psoriasis;cardiovascular disease; renal osteodystrophy; renal disease; end-stagerenal disease; chronic kidney disease; chronic kidney disease-associatedmineral and bone disorder; extraskeletal calcification; obesity;allergy, asthama; multiple sclerosis; muscle weakness; rheumatoidarthritis and diabetes.

In some embodiments, the invention can further comprise the step ofadministering a vitamin D insufficiency treatment to a subject who isdetermined to have a vitamin D insufficiency. In some embodiments, thetreatment can comprise administering a compound selected from the groupconsisting of: calcitriol; dihydrotachysterol; doxercalciferol;paricalcitol; cholecalciferol and ergocalciferol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the relationship between total and free 25-hydroxyvitaminD and lumbar spine bone mineral density. DBP-Bound, Free, andBioavailable 25-hydroxyvitamin D (25(OH)D) levels were calculated frommeasured total 25(OH)D and vitamin D binding protein (DBP) levels. Total25(OH)D and DBP-bound 25(OH)D were not associated with lumbar spine bonemineral density (BMD). Free 25(OH)D and bioavailable 25(OH) D werepositively correlated with lumbar spine BMD.

FIG. 2 depicts the relationship between total or bioavailable 25(OH)Dand serum calcium. Total levels of 25(OH)D demonstrated no associationwith serum calcium levels (corrected for albumin) while bioavailable25(OH)D levels were positively associated with serum calcium.

FIG. 3 depicts the relationship between total or bioavailable 25(OH)Dand PTH. After adjustment for age, gender, race, and survival status atone year, bioavailable 25(OH)D was significantly negatively associatedwith PTH levels, while total 25(OH)D demonstrated no association withPTH.

FIG. 4 depicts sample selection for Example 2. 25(OH)D and 1,25(OH)2Dwere previously measured as part of a case-control study within theArMORR cohort. Equal numbers of cases (subjects who died within theirfirst year on dialysis) and controls were randomly selected from eachracial group.

FIG. 5. Hypotheses tested in Example 4.

FIG. 6 is a diagram of an embodiment of a system for performing an assayfor determining the level of bioavailable or free vitamin D in a bloodsample obtained from a subject.

FIG. 7 is a diagram of an embodiment of a comparison module as describedherein.

FIG. 8 is a diagram of an embodiment of an operating system andapplications for a computing system as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention described herein include assays. Systems, andmethods of treatment which are based on the inventors' discovery thattotal serum levels of vitamin D, the currently used clinical parameter,correlate poorly with measures of health such as bone mineral densityand parathyroid hormone levels. The inventors have found that levels ofbioavailable and free vitamin D correlate better with the same measuresof health and are therefore more indicative of whether a subject hassufficient vitamin D levels. Described herein are assays for measuringbioavailable and free vitamin D and methods of treating subjects forvitamin D insufficiency.

Materials, procedures and considerations necessary to understand and usethe disclosed methods are described in the following, as areexperimental results and non-limiting examples that demonstrate andillustrate various embodiments of the methods and assays described.

DEFINITIONS

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and the include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. Similarly, the word or isintended to include and unless the context clearly indicates otherwise.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The abbreviation,“e.g.” is derived from the Latin exempli gratia, and is used herein toindicate a non-limiting example. Thus, the abbreviation “e.g.” issynonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); TheELISA guidebook (Methods in molecular biology 149) by Crowther J. R.(2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis,1979, Scientific Newsletters; Immunology by Werner Luttmann, publishedby Elsevier, 2006. Definitions of common terms in molecular biology canalso be found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

The terms “decrease,” “reduce,” “reduced”, “reduction”, “decrease,” and“inhibit” are all used herein generally to mean a decrease by astatistically significant amount relative to a reference. However, foravoidance of doubt, “reduce,” “reduction” or “decrease” or “inhibit”typically means a decrease by at least 10% as compared to a referencelevel and can include, for example, a decrease by at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, up to andincluding, for example, the complete absence of the given entity orparameter as compared to a reference level, or any decrease between10-99% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” or“promote” are all used herein to generally mean an increase by astatically significant amount; for the avoidance of any doubt, the terms“increased”, “increase” or “enhance” or “activate” or “promote” means anincrease of at least 10% as compared to a reference level, for examplean increase of at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% increase or any increase between 10-100% as compared toa reference level, or at least about a 2-fold, or at least about a3-fold, or at least about a 4-fold, or at least about a 5-fold or atleast about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level.

As used herein, the term “proteins” and “polypeptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide”, which are used interchangeably herein, refer to a polymerof protein amino acids, including modified amino acids (e.g.,phosphorylated, glycated, glycosylated, etc.) and amino acid analogs,regardless of its size or function. “Protein” and “polypeptide” areoften used in reference to relatively large polypeptides, whereas theterm “peptide” is often used in reference to small polypeptides, butusage of these terms in the art overlaps. The terms “protein” and“polypeptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary polypeptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrieras commonly used in the pharmaceutical industry.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient or is toxic to the subject, usethereof in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above. In certain embodiments, the subject is a mammal, e.g., aprimate, e.g., a human.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of vitaminD insufficiency. In addition, the methods described herein can be usedto treat domesticated animals and/or pets. A subject can be male orfemale. A subject can be one who has been previously diagnosed with oridentified as suffering from or having vitamin D insufficiency or one ormore diseases or conditions associated with a vitamin D insufficiency,and optionally, but need not have already undergone treatment forvitamin D insufficiency or the one or more diseases or conditionsassociated with a vitamin D insufficiency. A subject can also be one whohas been diagnosed with or identified as suffering from vitamin Dinsufficiency or one or more diseases or conditions associated with avitamin D insufficiency, but who shows improvements in known vitamin Dinsufficiency risk factors as a result of receiving one or moretreatments for vitamin D insufficiency or one or more diseases orconditions associated with a vitamin D insufficiency. Alternatively, asubject can also be one who has not been previously diagnosed as havingvitamin D insufficiency or one or more diseases or conditions associatedwith a vitamin D insufficiency. For example, a subject can be one whoexhibits one or more risk factors for vitamin insufficiency or one ormore diseases or conditions associated with a vitamin D insufficiency ora subject who does not exhibit vitamin D insufficiency risk factors.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a difference of at leasttwo standard deviations (2SD).

Vitamin D Insufficiency

Aspects of the invention described herein include assays directed todetermining whether a subject has a vitamin D insufficiency and methodsof treating these conditions. As used herein, “vitamin D” refers to anyof several forms of D vitamins including vitamin D1, D2, D3, D4 andisomers or derivatives thereof. Non-limiting examples of forms ofvitamin D include vitamin D2 (ergocalciferol) which is produced byplants, vitamin D3 (cholecalciferol) which is produced by animals. Bothvitamin D2 and D3 are hydroxylated in the liver to form, respectively,25(OH)D2 (25-hydroxyvitamin D2) and 25(OH)D3 (25-hydroxyvitamin D3 orcalcidiol), which can be referred to collectively as 25(OH)D. 25(OH)D isthe primary transport form of vitamin D in the body and is theprohormone of the active vitamin D hormones. Further hydroxylation,primarily in the kidneys, converts 25(OH)D to 1,25(OH)₂D (including1,25(OH)₂D3 (calcitriol) and 1,25(OH)₂D2). All of the foregoing forms ofvitamin D are encompassed by the term “vitamin D” as used herein. Insome embodiments, the level of vitamin D that is determined is the levelof 25-(OH)D. In some embodiments, the level of vitamin D that isdetermined is the level of 25-(OH)D2. In some embodiments, the level ofvitamin D that is determined is the level of 25-(OH)D3. In someembodiments, the level of vitamin D that is determined is the level of1,25-(OH)₂D. In some embodiments, the level of vitamin D that isdetermined is the level of 1,25-(OH)₂D2. In some embodiments, the levelof vitamin D that is determined is the level of 1,25-(OH)₂D3.

Vitamin D hormones influence bone mineralization and a number of aspectsof blood chemistry, including blood calcium levels and blood phosphoruslevels. Diseases and conditions which are caused by or associated withinsufficient levels of vitamin D are referred to herein as “vitaminD-associated diseases.” Insufficient levels of vitamin D are associatedwith secondary hyperparathyroidism, parathyroid gland hyperplasia,elevated parathyroid hormone levels. hypocalcemia, chronic kidneydisease (CKD), renal disease, end-stage renal disease, chronic kidneydisease-associated mineral and bone disorder, psoriasis, low bonemineral density, bone resorption and metabolic bone diseases such asfibrogenesis imperfecta ossium, osteitis fibrosa cystica, osteomalacia,rickets, osteoporosis, osteosclerosis, non-traumatic fractures of thespine and hip, renal osteodystrophy, and extraskeletal calcification.Secondary hyperparathyroidism (SHPT) increases bone turnover, and ifleft untreated, can impair mineralization and decrease bone mass.Patients with SHPT have increased bone turnover and decreased bone massthat can eventually progress to osteomalacia. Osteomalacia is a severedefect in or absence of bone mineralization occurring when both vitaminD and dietary calcium levels are markedly reduced. Osteoporosis, definedas a deficiency of normal bone within bone tissue, can result eitherfrom a low calcium diet with replete vitamin D levels or with lowvitamin D and adequate dietary calcium. Low serum 25(OH)D increases therisk of osteoporotic fractures, especially in older adults, and vitaminD and calcium supplementation at sufficient doses reduces the risk. Anumber of “non-classical” biologic effects have been reported forvitamin D beyond its “classical” effects on the parathyroid honnonesystem. Such effects have been reported in connection with cellulargrowth and differentiation, cellular proliferation, red blood cellformation, hair growth, muscular function, blood pressure, fibrosis, theimmune system and the cardiovascular system, including therenin-angiotensin system. Vitamin D insufficiency has been implicated inthe development or progression of, for example, infection,cardiovascular disease, allergy, asthama obesity, diabetes, muscleweakness, multiple sclerosis, rheumatoid arthritis and cancer.

Vitamin D insufficiency can be caused by insufficient exposure tosunlight, insufficient dietary intake of vitamin D, or conditions orclinical procedures, such as bariatric surgery, that result in reducedintestinal absorption of fat soluble vitamins such as vitamin D. VitaminD levels are traditionally measured as the level of total serum 25(OH)D.Although total serum 25(OH)D is currently the most widely accepted assayfor determining vitamin D status, it is subject to tremendous variationin results and interpretation, and may not be clinically relevant acrossall populations. Aspects of the invention described herein are directedto an assay for determining whether a subject is vitamin D insufficientby measuring the levels of bioavailable or free vitamin D, as opposed tototal serum vitamin D.

As used herein, “vitamin D insufficiency” refers to suboptimal levels ofvitamin D that can be associated with an increased risk of developingone or more of the conditions or diseases in which low vitamin D levelshave been implicated, which are described above herein. Subjects with avitamin D insufficiency may not have any symptoms, markers or signs of avitamin D-associated disease or may have symptoms, markers or signs ofone or more vitamin D-associated diseases. A subject who has a vitamin Dinsufficiency can be a subject who has a level of bioavailable or freevitamin D which is 25% or lower than the mean level of that form ofvitamin D measured in a healthy population of subjects. For example, asubject who has a level of bioavailable or free vitamin D which is 25%,or 20% or 15% or 10% or 5% or lower than the mean level of that form ofvitamin D in a population of healthy subjects has an insufficient levelof vitamin D.

A healthy subject can be one who does not display any markers, signs orsymptoms of a vitamin D-associated disease or condition and who is notat risk of having a vitamin D insufficiency. By way of non-limitingexample, a healthy subject will not exhibit signs or symptoms ofrickets, which include, for example delayed growth, pain in the spine,pelvis or legs, muscle weakness, or skeletal deformities such as bowedlegs, abnormal curvature of the spine, thickened wrists and ankles,and/or projection of the breastbone. Risk factors for vitamin Dinsufficiency are well-known in the art and can include, but are notlimited to, not drinking vitamin D fortified milk (e.g. lactoseintolerant subjects, subjects with milk allergies, some vegetarians, andbreast-fed infants); dark skin; old age (e.g. the elderly have a reducedability to synthesize vitamin D and can be more likely to stay indoors),chronic or acute or severe illness (conditions which make it likely thesubject will stay indoors, in hospitals, in intensive care facilities,or institutional and assisted-care facilities, including subjects withAlzheimer's disease or who are mentally ill); covering all exposed skin(such as members of certain religions or cultures); regular use ofsunscreen (e.g., the application of sunscreen with a Sun ProtectionFactor (SPF) value of 8 reduces production of vitamin D by 95%, andhigher SPF values may further reduce vitamin D); having or having beendiagnosed with a fat malabsorption syndrome (including but not limitedto cystic fibrosis, cholestatic liver disease, other liver disease,gallbladder disease, pancreatic enzyme deficiency, Crohn's disease,inflammatory bowel disease, sprue or celiac disease, or surgical removalof part or all of the stomach and/or intestines); having had small bowelresections; taking medications that increase the catabolism of vitaminD, including phenytoin, fosphenytoin, phenobarbital, carbamazepine, andrifampin; taking medications that reduce absorption of vitamin D,including cholestyramine, colestipol, orlistat, mineral oil, and fatsubstitutes; taking medications that inhibit activation of vitamin D,including ketoconazole; taking medications that decrease calciumabsorption, including corticosteroids; having or having been diagnosedas having gum disease, diabetes mellitus, insulin resistance syndrome,endothelial dysfunction (vitamin D deposited in body fat stores is lessbioavailable) cardiovascular disease, artherosclerosis heart failure orosteoporosis; being obese; or being a postmenopausal woman.

Bioavailable Vitamin D

The assays and methods of treatments described herein are based on theinventors' discovery that the level of bioavailable and/or free vitaminD in the blood of a subject has a more significant correlation tomeasures of health such as bone mineral density and parathyroid hormonelevels than does the level of total serum vitamin D.

In the blood stream, vitamin D can exist in one of three states; 1)bound by vitamin D binding protein, 2) bound by albumin protein or 3)unbound. As used herein, “vitamin D binding protein”, “VDBP” or “DBP”refers to a polypeptide of any of SEQ ID NO: 1, 2 or 3 (NCBI Gene ID No:2638) and naturally occurring variants (e.g. alleles), homologs andfunctional derivatives thereof. VDBP binds vitamin D tightly, with aK_(D)=0.7×10⁹ M⁻¹ (for human VDBP). The fraction of vitamin D which isbound to VDBP is referred to herein as “D_(VDBP)”, “D_(DBP)”, “VitaminD_(DBP)” or “Vitamin D_(VDBP).” As used herein, “albumin” refers to thepolypeptide of any of SEQ ID NO: 4, 5 or 6 (NCBI Gene ID No: 213) andnaturally occurring variants (e.g. alleles), homologs and functionalderivatives thereof. Albumin binds vitamin D less tightly than VDBP,with a K_(D)=6×10⁵ M⁻¹ (for human albumin). The fraction of vitamin Dwhich is bound to albumin is referred to herein as “D_(albumin)”,“D_(Alb)”, “vitamin D_(albumin)” or “vitamin D_(Alb).” Unbound vitamin Dis also referred to herein as “free vitamin D” or “D.” As used herein,“bioavailable vitamin D” refers to, collectively, free vitamin D andvitamin D bound to albumin. Bioavailable vitamin D does not comprise thefraction of vitamin D which is bound to VDBP. Bioavailable vitamin D isinterchangeably referred to herein as “V_(Bio)” and “Vitamin D_(Bio)”.

An Assay for Bioavailable Vitamin D

Aspects of the invention described herein are directed to assays todetermine the level of bioavailable and/or free vitamin D in a bloodsample obtained from a subject.

In some embodiments, the level of bioavailable and/or free vitamin D isdetermined by first determining the level of VDBP polypeptide, albuminpolypeptide and total vitamin D in a blood sample obtained from asubject. The level of free and bioavailable vitamin D can then becalculated using these values and the binding constants of VDBP andalbumin for vitamin D. For human proteins, the binding constants are,respectively, 0.7×10⁹ M⁻¹ and 6×10⁵ M⁻¹.

As used herein a “blood sample” refers to any amount of blood or afraction thereof that has been obtained from a subject. In someembodiments, the blood sample can comprise whole blood or a fractionthereof, e.g. serum or plasma. In some embodiments, the blood sample iscontacted with an anticoagulant or preservative prior to performing anassay as described herein. Non-limiting examples of anticoagulants andpreservatives include CPD, CP2D (Citrate Phosphate Double Dextrose),CPDA-1, CDP/ADSOL®, CDP/Optisol®, AS-3 (Additive Solution 3, HaemoneticsCorp Braintree Mass.) and SAG-M.

In some embodiments, a blood sample can be stored prior to being used inan assay as described herein. In some embodiments the blood sample canbe stored for any given period of time, e.g. minutes, hours, days,weeks, up to months, prior to use in an assay as described herein. Inone embodiment, the blood sample is frozen. In one embodiment, the bloodsample is not frozen.

In some embodiments, the assays described herein are performed on awhole blood sample. In some embodiments, the assays described herein areperformed on the plasma fraction of a blood sample. In some embodiments,the assays described herein are performed on the serum fraction of ablood sample.

The level of VDBP and/or albumin polypeptide present in the blood sampleobtained from a subject can be determined by any method for determiningthe level of a specific polypeptide known in the art. In someembodiments, the assay is performed on an automated analyzer.Non-limiting examples of methods that can be used in the methods andassays described herein include enzyme linked immunosorbent assay;dye-based photometric assay; western blot; immunoprecipitation;radioimmunological assay (RIA); radioimmunometric assay;chemiluminescent immunosorbent assay; electrochemiluminescentimmunosorbent assay; fluorescent immunosorbent assay; dye linkedimmunosorbent assay; immunoturbidimetric assay; immunonephelometricassay; immunofluorescence assay and mass spectroscopy. Various methodsof producing antibodies with a known antigen (e.g. a peptide comprisedby the polypeptides of SEQ ID Nos. 1-6) are well-known to thoseordinarily skilled in the art (see Antibodies: A Laboratory Manual(Harlow & Lane eds., 1988), which is hereby incorporated by reference inits entirety). In particular, suitable antibodies may be produced bychemical synthesis, by intracellular immunization (i.e., intrabodytechnology), or preferably, by recombinant expression techniques.Methods of producing antibodies may further include the hybridomatechnology well-known in the art. Antibodies specific for albumin andVDBP are commercially available (e.g. Cat. #ab112888 and ab23484,respectively, Abcam; Cambridge, Mass.).

In some embodiments, albumin levels can be determined by dye-basedphotometric assays on an automated analyzer. Dye-based photometricassays are commercially available (e.g. the Albumin FS™ kits; DiaSysDiagnostic Systems Gmb; Holzheim, Germany or the Albumin reagent, Cat#OSR6102; Beckman Coulter; Brea, Calif.). Automated analyzers arecommercially available (e.g. the AU2700 or AU5400 from Beckman Coulter;Brea, Calif.). Systems which are designed specifically for thedetermination of serum albumin levels are also available commercially(e.g. the Careside Analyzer™, Careside Inc., Culver City, Calif.). Insome embodiments, the level of albumin levels can be determined usingimmunoassays, e.g. the Human Serum Albumin ELISA Kit (Cat #1190; AlphaDiagnostic International; San Antonio, Tex.).

In some embodiments, VDBP levels can be determined by ELISA. ELISAassays for VDBP are commercially available (e.g. Cat #DVDBP0; R&DSystems; Minneapolis, Minn.). In some embodiments, the assay isconducted after diluting serum samples 1 to 2,000 in Calibrator DiluentRD6-11 (R&D Systems Part Number 895489). ELISA is a technique fordetecting and measuring the concentration of an antigen using a labeled(e.g. enzyme linked) form of the antibody. There are different forms ofELISA, which are well known to those skilled in the art. The standardtechniques known in the art for ELISA are described in “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc.,1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904;which are incorporated by reference herein in their entirety.

The level of total vitamin D present in the blood sample obtained from asubject can be determined by any method known in the art. Non-limitingexamples of methods that can be used in the methods and assays describedherein include radioimmunoassay; liquid chromatography tandem massspectroscopy; enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; and high-pressure liquidchromatography. In some embodiments, the level of total vitamin D in asample is determined by liquid chromatography tandem mass spectrometry(LC-MS). In some embodiments, the level of total vitamin D in a sampleis determined by high performance liquid chromatography/massspectrophotometry. In some embodiments, the level of total vitamin D ina sample is determined by radioimmunoassay. In some embodinents, thelevel of total vitamin D in a sample is determined using a commerciallyavailable radioimmunoassay (e.g. DiaSorin Inc, Stillwater, Minn., USA).In some embodiments, the level of total vitamin D in a sample isdetermined using a commercially available immunluminometric assay (e.g.Cat No 310600; DiaSorin Inc.; Stillwater MN.). The method of measuringtotal vitamin D in a blood sample obtained from a subject can alsoinclude liquid chromatography tandem mass spectroscopy as described inU.S. Pat. No. 7,700,365, which is included by reference herein in itsentirety.

Mass spectroscopy methods are well known in the art and have been usedto quantify and/or identify biomolecules. In some embodiments, thesignal strength of peak values from spectra of a first sample and asecond sample can be compared (e.g., visually, by computer analysisetc.), to determine the relative amounts of particular biomolecules.Software programs such as the Biomarker Wizard program (CiphergenBiosystems, Inc., Fremont, Calif.) can be used to aid in analyzing massspectra.

In some embodiments, the level of total vitamin D which is determinedcan comprise one or more forms of vitamin D selected from the groupconsisting of 25-hydroxyvitamin D (25(OH)D); 1,25-dihydroxyvitamin D(1,25-(OH)₂D); 25(OH)D2; 25(OH)D3; 1,25(OH)₂D2; 1,25-(OH)₂D3; vitaminD1; vitamin D2; vitamin D3; vitamin D4; ergocalciferol; cholecalciferol;calcidiol and calcitriol. In some embodiments, the level of totalvitamin D which is determined can comprise 25-hydroxyvitamin D(25(OH)D). In some embodiments, the level of total vitamin D which isdetermined can comprise 25-hydroxyvitamin D2 (25(OH)D2). In someembodiments, the level of total vitamin D which is determined cancomprise 25-hydroxyvitamin D3 (25(OH)D3). In some embodiments, the levelof total vitamin D which is determined can comprise1,25-dihydroxyvitamin D (1,25-(OH)₂D). In some embodiments, the level oftotal vitamin D which is determined can comprise 1,25-dihydroxyvitaminD2 (1,25-(OH)₂D2). In some embodiments, the level of total vitamin Dwhich is determined can comprise 1,25-dihydroxyvitamin D3(1,25-(OH)₂D3).

In some embodiments, once the levels of VDBP polypeptide, albuminpolypeptide and total vitamin D in a blood sample obtained from asubject are determined, the level of free and/or bioavailable vitamin Dcan be determined. The level of free vitamin D can be calculated usingEquation 8, the derivation of which is described in Example 1 herein.Free Vitamin D={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K_(alb) ·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K_(alb) ·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)}).  (Eq. 8)

The level of bioavailable vitamin D can be calculated using Equation 9,the derivation of which is described in Example 1 herein.Bioavailable Vitamin D=(K _(alb) ·[Alb]+1)·[Free Vitamin D]  (Eq. 9)

In some embodiments, the level of free (unbound) and/or bioavailablevitamin D can be determined directly. In some embodiments, the level offree vitamin D can be determined directly and used to calculate thelevel of bioavailable vitamin D. In some embodiments, the level of freevitamin D and the level of albumin polypeptide can be determineddirectly and used to calculate the level of bioavailable vitamin D. Asused herein “determined directly” refers to determining the level of afirst form of a vitamin or polypeptide by measuring or detecting thelevel of the first form of a vitamin or polypeptide as opposed tocalculating the level of the first form of a vitamin or polypeptideusing the level of a second or further form of a vitamin or polypeptidewhich was directly determined.

Direct measurement of the level of free and/or bioavailable vitamin Dcan be accomplished by a number of methods. Non-limiting examples ofmethods for direct measurement of the level of free vitamin D includethe following. 1) Vitamin D complexed with VDBP can be depleted from asample using immunodepletion or affinity binding chromatography todeplete VDPB from the sample. The remaining vitamin D (the bioavailablefraction) can then be measured according to any of the methods describedelsewhere herein. 2) Vitamin D complexed with VDBP can be depleted froma sample using differential precipitation of VDBP with antibodies(immunoprecipitation), actin (affinity precipitation) with or withoutprecipitating buffers (e.g. polyethylene glycol, ammonium sulfate)followed by centrifugal separation from free and/or bioavailable vitaminD and measurement of remaining vitamin D fraction according to any ofthe methods described elsewhere herein. 3) Immunoassays can also be used(see for example, Ekins et al. J Endocrinol Invest. 9 Suppl 4:3-15.1986; which is incorporated by reference herein in its entirety).Immunoassays capitalize on the idea that free analyte may be measuredusing lower affinity antibodies which do not “strip” the vitamin fromits high affinity binding protein. Free and/or bioavailable vitamin Dcan be measured directly using several competitive immunoassayapproaches described below and in (Christofides, Nic D. The ImmunoassayHandbook, 3^(rd) Ed. Editor David Wild, Elsevier Ltd, 2005; which isincorporated by reference herein in its entirety) These methods are allbased upon two principals: (1) Antibodies will differentiate betweenfree and protein-bound analytes if the reaction conditions (pH,temperature, buffers) do not interfere with binding between the vitaminand its binding protein, and (2) The total antibody binding capacity(affinity constant times antibody concentration) does not significantlydeplete the total vitamin D concentration and thus does not “strip” theprotein-bound vitamin D. This may be achieved using an antibody withrelatively low affinity (˜10¹⁰ L/M) for the vitamin D ligand and/orlimited assay reaction times which allow for binding of free andalbumin-bound vitamin D but are too short to allow dissociation ofVDBP-bound vitamin D.

Specific immunoassay designs that can be used include the following: A)Two-step measurement of free and/or bioavailable vitamin D with antibodycapture—(1) capture of free and/or bioavailable vitamin D withimmobilized vitamin D-binding antibody, (2) wash away unbound vitamin Dand VDBP, (3) detection of bound vitamin D by competitive binding withlabeled vitamin D (or labeled vitamin D analog that also binds theantibody). B) One-step measurement of free and/or bioavailable vitamin Dwith immobilized vitamin D-binding antibody and competitive detectionusing labeled vitamin D (or labeled vitamin D analog). C) One-stepmeasurement of free and/or bioavailable vitamin D with immobilizedvitamin D or vitamin D analog and labeled vitamin D-binding antibody.Free and/or bioavailable vitamin D from a sample competes with theimmobilized vitamin D for binding to the labeled antibody. D) One-stepmeasurement of free and/or bioavailable vitamin D with fluorescencepolarization immunoassay (Mendel C M. Clin Chem. 38(9):1916-7. 1992;which is incorporated by reference herein in its entirety). Free and/orbioavailable vitamin D and fluorescently labeled vitamin D analogcompete for binding to antibody and polarized fluorescence indicatesrelative amount of free and/or bioavailable vitamin D competing forbinding sites.

In some embodiments, determining of the level of free and/or vitamin Dcan comprise the use of a method selected from the group consisting of:immunoassay; two-step immunoassay with antibody capture; one-stepimmunoassay with immobilized antibody and competitive detection;one-step immunoassay with immobilized competitior and labeled antibody;fluorescence polarization immunoassay; differential precipitation(immunoprecipitation, affinity precipitation); immunodepletion; andaffinity binding chromatography; and a method selected from the groupconsisting of: radioimmunoassay; chemiluminescent immunosorbent assay;electrochemiluminescent immunosorbent assay; fluorescent immunosorbentassay; dye linked immunosorbent assay; liquid chromatography tandem massspectroscopy and high-pressure liquid chromatography.

In some embodiments, when the level of bioavailable or free vitamin Ddetermined to be in the blood sample of a subject is less than 25% ofthe mean value of bioavailable or free vitamin D in a population ofhealthy subjects, the subject is likely to have a vitamin Dinsufficiency. In some embodiments, when the level of bioavailable orfree vitamin D determined to be in the blood sample of a subject is lessthan 25% of the mean value of bioavailable or free vitamin D in apopulation of healthy subjects, the subject is indicated to have avitamin D insufficiency. In some embodiments, when the level ofbioavailable or free vitamin D determined to be in the blood sample of asubject is less than 25% of the mean value of bioavailable or freevitamin D in a population of healthy subjects, the subject has anincreased likelihood of having or developing a vitamin D-associateddisease. In some embodiments, when the level of bioavailable or freevitamin D determined to be in the blood sample of a subject is less than25% of the mean value of bioavailable or free vitamin D in a populationof healthy subjects, the subject is in need of a treatment for vitamin Dinsufficiency. In some embodiments, when the level of bioavailable orfree vitamin D determined to be in the blood sample of a subject is lessthan 25% of the mean value of bioavailable or free vitamin D in apopulation of healthy subjects, the subject has a greater likelihood ofbeing in need of a treatment for vitamin D insufficiency.

In some embodiments, when the level of bioavailable or free vitamin Ddetermined to be in the blood sample of a subject is more than 25% ofthe mean value of bioavailable or free vitamin D in a population ofhealthy subjects, the subject is not treated for vitamin Dinsufficiency.

Methods of Treating Vitamin D Insufficiency

Aspects of the invention described herein are directed to methods oftreating a vitamin D insufficiency comprising detecting the level ofbioavailable or free vitamin D in a blood sample obtained from a subjectand administering a treatment for vitamin D insufficiency if the levelof bioavailable vitamin D is below 25% of the mean value of bioavailablevitamin D in a population of healthy subjects. In some embodiments, thelevel of bioavailable or free vitamin D is determined by determining thelevel of VDBP polypeptide, albumin polypeptide and total vitamin D in ablood sample obtained from a subject and calculating the level of freeand/or bioavailable vitamin D as described above herein. In someembodiments, the level of free (unbound) and/or bioavailable vitamin Dcan be determined directly. In some embodiments, the level of freevitamin D can be determined directly and used to calculate the level ofbioavailable vitamin D. In some embodiments, the level of free vitamin Dand the level of albumin polypeptide can be determined directly and usedto calculate the level of bioavailable vitamin D.

In some embodiments, a treatment for vitamin D insufficiency caninclude, for example, compounds which increase the level of vitamin D,bioavailable vitamin D and/or free vitamin D in the subject by providingone or more forms of vitamin D, stimulating the endogenous production ofvitamin D, stimulating the production of active forms of vitamin Dand/or inhibiting the metabolism of vitamin D. Many naturally-occurringforms of vitamin D, derivatives and analogs thereof can be administeredto subjects in need of a vitamin D insufficiency treatment. In someembodiments, any form of vitamin D or a derivative or analog thereof maybe used provided that it exhibits one or more activities ofnaturally-occurring active vitamin D (e.g. increases intestinal calciumabsorption, serum calcium levels or bone mineralization) or ismetabolized to a compound that exhibits such activity. Non-limitingexamples of such compounds include alfacalcidol; calcifediol;calcipotriene; calcidiol; calcitriol (Rocaltrol; Roche);dihydrotachysterol (DHT™ and DHT Intensol™; Roxane Laboratories);doxercalciferol (Hectorol®; Genzyme); paricalcitol (Zemplar®; AbbottLaboratories); cholecalciferol (Delta D3™; Freeda Vitamins Inc.) andergocalciferol (Drisdol; Sanofi). Cholecalciferol and ergocalciferol areavailable as dietary supplements. Further non-limiting examples include5,6-trans-cholecalciferols; 5,6-trans-ergocalciferols; fluorinatedcholecalciferols; side chain homologated cholecalciferols; sidechain-truncated cholecalciferols; 19-nor cholecalciferols andergocalciferols; 10,19-dihydovitamin D compounds; 25-hydroxyvitamin D3;25-hydroxyvitamin D2; 24, 24-difluoro-25-hydroxyvitamin D3;24-fluoro-25-hydroxyvitamin D3;26,26,26,27,27,27hexafluoro-25-hydroxyvitamin D3; 24-25-dihydroxyvitaminD3; d5,26dihydroxyvitamin D3; 23,25,26-trihydroxyvitamin D3;25-hydroxyvitamin D3; the side chain, nor, dinor, trinor andtetranoranalogs of 25-hydroxyvitamin D3, 24-homo-1,25-dihydroxyvitaminD3; 24-dihomo-1,25-dihydroxyvitamin D3; 24-trihomo-1,25-dihydroxyvitaminD3; as well as the corresponding 19-nor compounds of those listed above.

Vitamin D activity can be assayed by a number of methods known in theart. A non-limiting example of an assay to determine if a compound hasvitamin D activity or is metabolized in the subject's body to a compoundhaving vitamin D activity is described in U.S. Pat. No. 5,532,229 whichis incorporated by reference herein in its entirety. Briefly, thecompound is administered and the levels of serum calcium are determinedby chemical colorimetry or by treating with nitric acid and measuringatomic absorption. Administration of a compound having vitamin Dactivity or that is metabolized to a compound having vitamin D activitywill increase the serum calcium levels. Other non-limiting examples ofassays for vitamin D activity include bone mineral density as measuredby x-ray absorptiometry (DEXA) or measurement of serum osteocalcin (seeU.S. Pat. No. 5,972,917 which is incorporated by reference herein in itsentirety).

The dosage of a treatment for vitamin D insufficiency can be determinedby a physician and adjusted, as necessary, to suit observed effects ofthe treatment. With respect to duration and frequency of treatment, itis typical for skilled clinicians to monitor subjects in order todetermine when the treatment is providing therapeutic benefit, and todetermine whether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment ormake other alteration to treatment regimen.

The dosage ranges for the administration of a treatment for vitamin Dinsufficiency depend upon the form of the treatment for vitamin Dinsufficiency, and its potency, as described further herein, and areamounts large enough to produce the desired effect in which thesymptoms, markers, or signs of vitamin D insufficiency are reduced. Insome embodiments, the symptoms, markers, or signs of vitamin Dinsufficiency can include the level of bioavailable or free vitamin Ddetermined according to the methods described herein. The dosage shouldnot be so large as to cause substantial adverse side effects. Generally,the dosage can vary with the age, condition, and sex of the patient andcan be determined by one of ordinary skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication or based upon the subject's sensitivity to the treatment.By way of non-limiting example, forms of vitamin D or a derivative oranalog thereof are typically administered in a therapeutically effectiveamount of from about 0.1 μg to about 2 mg per day depending upon thecompound being administered.

In some embodiments, a vitamin D insufficiency treatment can beadministered over a period of time, such as over a 5 minute, 10 minute,15 minute, 20 minute, or 25 minute period. In some embodiments, theadministration can be repeated, for example, on a regular basis, such ashourly for 3 hours, 6 hours, 12 hours or daily or longer or such asbiweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. In some embodiments, when multiple dosesare administered, the doses can be separated from one another by, forexample, six hours, one day, two days, one week, two weeks, one month,or two months.

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration biweekly forthree months, administration can be repeated once per month, for sixmonths or a year or longer. In some embodiments, administration can bechronic, e.g., one or more doses daily over a period of weeks or months.

Administration of a treatment for vitamin D insufficiency can reducelevels of a marker or symptom of vitamin D insufficiency or a disease orcondition associated with vitamin D insufficiency by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% ormore. As used herein, the phrase “therapeutically effective amount”,“effective amount” or “effective dose” refers to an amount that providesa therapeutic benefit in the treatment or management of vitamin Dinsufficiency. Vitamin D insufficiency can be determined according tothe methods described herein and a therapeutically effective amount canbe an amount that provides a statistically significant improvement inthe level of bioavailable or free vitamin D as determined according tothe methods described herein. Determination of a therapeuticallyeffective amount is well within the capability of those of ordinaryskill in the art. Generally, a therapeutically effective amount can varywith the subject's history, age, condition, and gender, as well as theseverity and type of the medical condition in the subject, andadministration of other pharmaceutically active agents.

In some embodiments, the administration is repeated until the level ofbioavailable or free vitamin D, as determined according to the methodsdescribed herein, no longer indicates that the subject is vitamin Dinsufficient.

It is to be understood that, for any particular subject, specific dosageregimes should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions. For example, the dosage of thetherapeutic can be increased if the lower dose does not providesufficient therapeutic activity.

Alternative methods of treating a subject for a vitamin D insufficiencycan include, by way of non-limiting example, exposure to sunlight oradministration of a CYP24 inhibitor (see U.S. patent application Ser.No. 12/935,139); dentonin peptides (see U.S. patent application Ser. No.10/360,202); IL-10 or IL-4 polypeptides or TNF-α inhibitors (see U.S.patent application Ser. No. 10/170,746); or PHEX polypeptides asdescribed in U.S. patent application Ser. No. 10/360,202.

With respect to the therapeutic methods of the invention, unlessotherwise specified, it is not intended that the administration of thevitamin D insufficiency treatment be limited to a particular mode ofadministration, dosage, or frequency of dosing; the present inventioncontemplates all modes of administration, including intramuscular,intravenous, intraperitoneal, intravesicular, intraarticular, topically,subcutaneous, orally or any other route sufficient to provide a doseadequate to treat the vitamin D insufficiency.

Systems

In some aspects, the invention described herein is directed to systems(and computer readable media for causing computer systems) for obtainingdata from at least one blood sample obtained from at least one subject,the system comprising 1) a determination module configured to receivethe at least one blood sample and perform at least one analysis on theat least one blood sample to determine the level of bioavailable or freevitamin D in the sample; 2) a storage device configured to store dataoutput from the determination module; and 3) a display module fordisplaying a content based in part on the data output from thedetermination module, wherein the content comprises a signal indicativeof the level of bioavailable or free vitamin D.

In one embodiment, provided herein is a system comprising: (a) at leastone memory containing at least one computer program adapted to controlthe operation of the computer system to implement a method that includes(i) a determination module configured to receive the at least one bloodsample and perform at least one analysis on the at least one bloodsample to determine the level of bioavailable or free vitamin D in thesample (e.g. determining the level of one or more of VDBP polypeptide,albumin polypeptide, total vitamin D; bioavailable vitamin D; and freevitamin D); (ii) a storage module configured to store output data fromthe determination module; (iii) a computing module adapted to identifyfrom the output data whether the level of VDBP polypeptide, albuminpolypeptide, total vitamin D, bioavailable vitamin D or free vitamin Din a blood sample obtained from a subject indicates that the level ofbioavailable or free vitamin D is lower than 25% of the mean value ofbioavailable or free vitamin D in a population of healthy subjects and(iv) a display module for displaying a content based in part on the dataoutput from the determination module, wherein the content comprises asignal indicative of the level of bioavailable or free vitamin D and (b)at least one processor for executing the computer program (see FIG. 6).

The term “computer” can refer to any non-human apparatus that is capableof accepting a structured input, processing the structured inputaccording to prescribed rules, and producing results of the processingas output. Examples of a computer include: a computer; a general purposecomputer; a supercomputer; a mainframe; a super mini-computer; amini-computer; a workstation; a micro-computer; a server; an interactivetelevision; a hybrid combination of a computer and an interactivetelevision; and application-specific hardware to emulate a computerand/or software. A computer can have a single processor or multipleprocessors, which can operate in parallel and/or not in parallel. Acomputer also refers to two or more computers connected together via anetwork for transmitting or receiving information between the computers.An example of such a computer includes a distributed computer system forprocessing information via computers linked by a network.

The term “computer-readable medium” may refer to any storage device usedfor storing data accessible by a computer, as well as any other meansfor providing access to data by a computer. Examples of astorage-device-type computer-readable medium include: a magnetic harddisk; a floppy disk; an optical disk, such as a CD-ROM and a DVD; amagnetic tape; a memory chip.

The term a “computer system” may refer to a system having a computer,where the computer comprises a computer-readable medium embodyingsoftware to operate the computer.

The term “software” is used interchangeably herein with “program” andrefers to prescribed rules to operate a computer. Examples of softwareinclude: software; code segments; instructions; computer programs; andprogrammed logic.

The computer readable storage media can be any available tangible mediathat can be accessed by a computer. Computer readable storage mediaincludes volatile and nonvolatile, removable and non-removable tangiblemedia implemented in any method or technology for storage of informationsuch as computer readable instructions, data structures, program modulesor other data. Computer readable storage media includes, but is notlimited to, RAM (random access memory), ROM (read only memory), EPROM(erasable programmable read only memory), EEPROM (electrically erasableprogrammable read only memory), flash memory or other memory technology,CD-ROM (compact disc read only memory), DVDs (digital versatile disks)or other optical storage media, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage media, other types ofvolatile and non-volatile memory, and any other tangible medium whichcan be used to store the desired information and which can accessed by acomputer including and any suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programsthat, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable storage medium describedherein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

Embodiments of the invention can be described through functionalmodules, which are defined by computer executable instructions recordedon computer readable media and which cause a computer to perform methodsteps when executed. The modules are segregated by function for the sakeof clarity. However, it should be understood that the modules/systemsneed not correspond to discreet blocks of code and the describedfunctions can be carried out by the execution of various code portionsstored on various media and executed at various times. Furthermore, itshould be appreciated that the modules can perform other functions, thusthe modules are not limited to having any particular functions or set offunctions.

The functional modules of certain embodiments of the invention includeat minimum a determination module, a storage module, a computing module,and a display module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The determination module has computer executable instructions to providee.g., levels of expression products etc in computer readable form.

The determination module can comprise any system for detecting a signalelicited from an assay to determine the level of any of a VDBPpolypeptide, an albumin polypeptide, bioavailable vitamin D, freevitamin D, or total vitamin D as described above herein. In someembodiments, such systems can include an instrument, e.g., AU2700(Beckman Coulter Brea, Calif.) as described herein for quantitativemeasurement of polypeptides. In another embodiment, the determinationmodule can comprise multiple units for different functions, such asquantitative measurement of polypeptides (e.g. dye-based photometricassay or quantitative ELISA) and a mass spectroscopy system for themeasurement of vitamin D. In one embodiment, the determination modulecan be configured to perform the methods described elsewhere herein,e.g. dye-based photometric assays for albumin, ELISA assays for VDBPpolypeptide levels or mass spectroscopy to determine vitamin D levels.In some embodiments, such systems can include an instrument, e.g., theAU2700 (Beckman Coulter Brea, Calif.).

In some embodiments, the determination system or a further module can beconfigured to process whole blood samples, e.g. to separate serum fromwhole blood for use in the assays described herein.

The information determined in the determination system can be read bythe storage module. As used herein the “storage module” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus, data telecommunications networks,including local area networks (LAN), wide area networks (WAN), Internet,Intranet, and Extranet, and local and distributed computer processingsystems. Storage modules also include, but are not limited to: magneticstorage media, such as floppy discs, hard disc storage media, magnetictape, optical storage media such as CD-ROM, DVD, electronic storagemedia such as RAM, ROM, EPROM, EEPROM and the like, general hard disksand hybrids of these categories such as magnetic/optical storage media.The storage module is adapted or configured for having recorded thereon,for example, sample name, biomolecule assayed and the level of saidbiomolecule. Such information may be provided in digital form that canbe transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage module. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising expression level information.

In some embodiments of any of the systems described herein, the storagemodule stores the output data from the determination module. Inadditional embodiments, the storage module stores reference informationsuch as levels of bioavailable or free vitamin D in healthy subjectsand/or a population of healthy subjects.

The “computing module” can use a variety of available software programsand formats for computing the level of bioavailable or free vitamin D.Such algorithms are well established in the art. A skilled artisan isreadily able to determine the appropriate algorithms based on the sizeand quality of the sample and type of data. The data analysis tools andequations described herein can be implemented in the computing module ofthe invention. In one embodiment, the computing module further comprisesa comparison module, which compares the level of bioavailable or freevitamin D in a blood sample obtained from a subject as described hereinwith the mean value of bioavailable or free vitamin D in a population ofhealthy subjects (FIG. 7). By way of an example, when the value ofbioavaible vitamin D in a blood sample obtained from a subject ismeasured, a comparison module can compare or match the output data—withthe mean value of bioavailable vitamin D in a population of healthysubjects. In certain embodiments, the mean value of bioavailable or freevitamin D in a population of healthy subjects can be pre-stored in thestorage module. During the comparison or matching process, thecomparison module can determine whether the level of bioavailable orfree vitamin D in the blood sample obtained from a subject is less than25% of the mean value of bioavailable or free vitamin D in a populationof healthy subjects. In various embodiments, the comparison module canbe configured using existing commercially-available or freely-availablesoftware for comparison purpose, and may be optimized for particulardata comparisons that are conducted.

The computing and/or comparison module, or any other module of theinvention, can include an operating system (e.g., UNIX) on which runs arelational database management system, a World Wide Web application, anda World Wide Web server. World Wide Web application includes theexecutable code necessary for generation of database language statements(e.g., Structured Query Language (SQL) statements). Generally, theexecutables will include embedded SQL statements. In addition, the WorldWide Web application may include a configuration file which containspointers and addresses to the various software entities that comprisethe server as well as the various external and internal databases whichmust be accessed to service user requests. The Configuration file alsodirects requests for server resources to the appropriate hardware—as maybe necessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). In someembodiments users can directly access data (via Hypertext links forexample) residing on Internet databases using a HTML interface providedby Web browsers and Web servers (FIG. 8).

The computing and/or comparison module provides a computer readablecomparison result that can be processed in computer readable form bypredefined criteria, or criteria defined by a user, to provide contentbased in part on the comparison result that may be stored and output asrequested by a user using an output module, e.g., a display module.

In some embodiments, the content displayed on the display module can bethe level of bioavailable or free vitamin D in the blood sample obtainedfrom a subject. In some embodiments, the content displayed on thedisplay module can be the relative level of bioavailable or free vitaminD in the blood sample obtained from a subject as compared to the meanlevel of bioavailable or free vitamin D in a population of healthysubjects. In some embodiments, the content displayed on the displaymodule can indicate whether the level of bioavailable or free vitamin Din the blood sample obtained from a subject is less or more than 25% ofthe mean value of bioavailable or free vitamin D in a population ofhealthy subjects. In some embodiments, the content displayed on thedisplay module can indicate whether the subject has an insufficientlevel of vitamin D. In some embodiments, the content displayed on thedisplay module can indicate whether the subject is in need of atreatment for vitamin D insufficieny. In some embodiments, the contentdisplayed on the display module can indicate whether the subject has anincreased risk or likelihood of having or developing a vitaminD-associated disease. In some embodiments, the content displayed on thedisplay module can be a numerical value indicating one of these risks orprobabilities. In such embodiments, the probability can be expressed inpercentages or a fraction. For example, higher percentage or a fractioncloser to 1 indicates a higher likelihood of a subject having a vitaminD-associated disease. In some embodiments, the content displayed on thedisplay module can be single word or phrases to qualitatively indicate arisk or probability. For example, a word “unlikely” can be used toindicate a lower risk for having or developing a vitamin D-associateddisease, while “likely” can be used to indicate a high risk for havingor developing a vitamin D-associated disease.

In one embodiment of the invention, the content based on the computingand/or comparison result is displayed on a computer monitor. In oneembodiment of the invention, the content based on the computing and/orcomparison result is displayed through printable media. The displaymodule can be any suitable device configured to receive from a computerand display computer readable information to a user. Non-limitingexamples include, for example, general-purpose computers such as thosebased on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., or anyother type of processor, visual display devices such as flat paneldisplays, cathode ray tubes and the like, as well as computer printersof various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the computing/comparisonresult. It should be understood that other modules of the invention canbe adapted to have a web browser interface. Through the Web browser, auser can construct requests for retrieving data from thecomputing/comparison module. Thus, the user will typically point andclick to user interface elements such as buttons, pull down menus,scroll bars and the like conventionally employed in graphical userinterfaces.

In some embodiments, the system further comprises a means of inputting avalue for the level of one or more of VDBP polypeptide, albuminpolypeptide, and total vitamin D determined to be in a blood sampleobtained from a subject. By way of non-limiting example, the level ofalbumin polypeptide can be determined by the determination module of thesystem while the level of VDBP polypeptide is determined by an ELISAassay performed separately from the system described herein. When thelevel of VDBP polypeptide is determined, the value for this level can beentered into the computing module of the system and used to determinethe level of bioavailable or free vitamin D in the blood sample obtainedfrom the subject. In some embodiments, the inputting means comprises akeyboard or touchscreen which allows a user to type a value which isaccepted by the computing module.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for determining the level ofbioavailable or free vitamin D in a blood sample obtained from asubject, and therefore are not intended to limit the scope of theinvention. Variations of the systems and computer readable mediadescribed herein are possible and are intended to fall within the scopeof the invention.

The modules of the machine, or those used in the computer readablemedium, may assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments can perform functions in a different order, or functions canbe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. These and other changes can be made to the disclosure inlight of the detailed description.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentscan also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs:

-   -   I. An assay comprising:        -   analyzing a blood sample obtained from a subject to            determine a level of VDBP (vitamin D binding protein)            polypeptide, albumin polypeptide and total vitamin D;        -   wherein a level of bioavailable vitamin D is:            =(K _(alb) *[Alb]+1)*[Free Vitamin D]        -   and wherein a level of free vitamin D is:            ={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)            ·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin            D]+K _(alb) ·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K            _(DBP))·([Total Vitamin D])}}÷(2·{K _(DBP) ·K _(alb)            ·[Alb]+K _(DBP)}).    -   II. The assay of paragraph I, wherein a level of bioavailable        vitamin D lower than 25% of the mean value of bioavailable        vitamin D in a population of healthy subjects indicates that the        subject has a vitamin D insufficiency.    -   III. The assay of any of paragraphs I-II, wherein the vitamin D        is selected from the group consisting of:        -   25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.    -   IV. The assay of any of paragraphs I-III, wherein the        determining of the level of VDBP polypeptide or albumin        polypeptide comprises use of a method selected from the group        consisting of:        -   enzyme linked immunosorbent assay; chemiluminescent            immunosorbent assay; electrochemiluminescent immunosorbent            assay; fluorescent immunosorbent assay; dye linked            immunosorbent assay; immunoturbidimetric assay;            immunonephelometric assay; dye-based photometric assay;            western blot; immunoprecipitation; radioimmunological assay            (RIA); radioimmunometric assay; immunofluorescence assay and            mass spectroscopy.    -   V. The assay of any of paragraphs I-IV, wherein the determining        of the level of total vitamin D comprises the use of a method        selected from the group consisting of:        -   radioimmunoassay; liquid chromatography tandem mass            spectroscopy; enzyme linked immunosorbent assay;            chemiluminescent immunosorbent assay;            electrochemiluminescent immunosorbent assay; fluorescent            immunosorbent assay; and high-pressure liquid            chromatography.    -   VI. The assay of any of paragraphs I-V, wherein an insufficiency        of vitamin D indicates an increased risk of a condition selected        from the group consisting of:        -   decreased bone density; decreased bone mineral density; bone            fractures; bone resorption; rickets; osteitis fibrosa            cystica; fibrogenesis imperfect ossium; osteosclerosis;            osteoporosis; osteomalacia; elevated parathyroid hormone            levels; parathyroid gland hyperplasia; secondary            hyperparathyroidism; hypocalcemia; infection; cancer;            psoriasis; cardiovascular disease; renal osteodystrophy;            renal disease; end-stage renal disease; chronic kidney            disease; chronic kidney disease-associated mineral and bone            disorder; extraskeletal calcification; obesity; allergy,            asthama; multiple sclerosis; muscle weakness; rheumatoid            arthritis and diabetes.    -   VII. The assay of any of paragraphs I-VI, further comprising the        step of administering a vitamin D insufficiency treatment to a        subject who is determined to have a vitamin D insufficiency.    -   VIII. The assay of any of paragraphs I-VII, wherein the        treatment comprises administering a compound selected from the        group consisting of:        -   calcitriol; dihydrotachysterol; doxercalciferol;            paricalcitol; cholecalciferol and ergocalciferol.    -   IX. An assay comprising:        -   analyzing a blood sample obtained from a subject to            determine a level of VDBP polypeptide, albumin polypeptide            and total vitamin D;        -   wherein a level of free vitamin D is:            ={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)            ·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin            D]+K _(alb) ·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K            _(DBP))·([Total Vitamin D])}}÷(2·{K _(DBP) ·K _(alb)            ·[Alb]+K _(DBP)}).    -   X. The assay of paragraph IX, wherein a level of free vitamin D        lower than 25% of the mean value of free vitamin D in a        population of healthy subjects indicates that the subject has a        vitamin D insufficiency.    -   XI. The assay of any of paragraphs IX-X, wherein the vitamin D        is selected from the group consisting of:        -   25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.    -   XII. The assay of any of paragraphs IX-XI, wherein the        determining of the level of VDBP polypeptide or albumin        polypeptide comprises the use of a method selected from the        group consisting of:        -   enzyme linked immunosorbent assay; chemiluminescent            immunosorbent assay; electrochemiluminescent immunosorbent            assay; fluorescent immunosorbent assay; dye linked            immunosorbent assay; immunoturbidimetric assay;            immunonephelometric assay; dye-based photometric assay;            western blot; immunoprecipitation; radioimmunological assay            (RIA); radioimmunometric assay; immunofluorescence assay and            mass spectroscopy.    -   XIII. The assay of any of paragraphs IX-XII, wherein the        determining of the level of total vitamin D comprises the use of        a method selected from the group consisting of:        -   radioimmunoassay; liquid chromatography tandem mass            spectroscopy; enzyme linked immunosorbent assay;            chemiluminescent immunosorbent assay;            electrochemiluminescent immunosorbent assay; fluorescent            immunosorbent assay; and high-pressure liquid            chromatography.    -   XIV. The assay of any of paragraphs IX-XIII, wherein an        insufficiency of vitamin D indicates an increased risk of a        condition selected from the group consisting of:        -   decreased bone density; decreased bone mineral density; bone            fractures; bone resorption; rickets; osteitis fibrosa            cystica; fibrogenesis imperfect ossium; osteosclerosis;            osteoporosis; osteomalacia; elevated parathyroid hormone            levels; parathyroid gland hyperplasia; secondary            hyperparathyroidism; hypocalcemia; infection; cancer;            psoriasis; cardiovascular disease; renal osteodystrophy;            renal disease; end-stage renal disease; chronic kidney            disease; chronic kidney disease-associated mineral and bone            disorder; extraskeletal calcification; obesity; allergy,            asthama; multiple sclerosis; muscle weakness; rheumatoid            arthritis and diabetes.    -   XV. The assay of any of paragraphs IX-XIV, further comprising        the step of administering a vitamin D insufficiency treatment to        a subject who is determined to have a vitamin D insufficiency.    -   XVI. The assay of any of paragraphs IX-XV, wherein the treatment        comprises administering a compound selected from the group        consisting of:        -   calcitriol; dihydrotachysterol; doxercalciferol;            paricalcitol; cholecalciferol and ergocalciferol.    -   XVII. A method for treating a vitamin D insufficiency in a        subject comprising detecting a level of VDBP polypeptide,        albumin polypeptide and total vitamin D in a blood sample        obtained from a subject;        -   wherein a level of bioavailable vitamin D is:            =(K _(alb) *[Alb]+1)*[Free Vitamin D]        -   and wherein a level of free vitamin D is:            ={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)            ·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin            D]+K _(alb) ·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K            _(DBP))·([Total Vitamin D])}}÷(2·{K _(DBP) ·K _(alb)            ·[Alb]+K _(DBP)})        -   and administering a treatment for vitamin D insufficiency to            the subject if the level of bioavailable vitamin D is less            than 25% of the mean value of bioavailable vitamin D in a            population of healthy subjects.    -   XVIII. A method for treating a vitamin D insufficiency in a        subject comprising detecting a level of VDBP polypeptide,        albumin polypeptide and total vitamin D in a blood sample        obtained from a subject;        -   wherein a level of free vitamin D is:            ={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)            ·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin            D]+K _(alb) ·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K            _(DBP))·([Total Vitamin D])}}÷(2·{K _(DBP) ·K _(alb)            ·[Alb]+K _(DBP)})        -   and administering a treatment for vitamin D insufficiency to            the subject if the level of free vitamin D is less than 25%            of the mean value of free vitamin D in a population of            healthy subjects.    -   XIX. A system for obtaining data from at least one blood sample        obtained from at least one subject, the system comprising:        -   a determination module configured to receive the at least            one blood sample and perform at least one analysis on the at            least one blood sample to determine a level of bioavailable            or free vitamin D in the sample;        -   a storage device configured to store data output from said            determination module; and        -   a display module for displaying a content based in part on            the data output from said determination module, wherein the            content comprises a signal indicative of the level of            bioavailable or free vitamin D.    -   XX. The system of paragraph XIX, wherein the system further        comprises a means of inputting a value for the level of one or        more of VDBP polypeptide, albumin polypeptide, and total vitamin        D determined to be in a test sample.    -   XXI. The system of any of paragraphs XIX-XX, wherein the content        displayed on said display module further comprises a signal        indicative of the subject having an increased likelihood of a        vitamin D insufficiency if the level of bioavailable or free        vitamin D is determined to be lower than 25% of the mean value        of bioavailable vitamin D in a population of healthy subjects.    -   XXII. The system of any of paragraphs XIX-XXI, wherein the        content displayed on said display module further comprises a        signal indicative of the subject being recommended to receive a        treatment for vitamin D insufficiency.    -   XXIII. A method of treatment comprising:        -   analyzing a blood sample obtained from a subject to            determine a level of free or bioavailable vitamin D;        -   wherein a level of free or bioavailable vitamin D lower than            25% of the mean value of free or bioavailable vitamin D in a            population of healthy subjects indicates that the subject            has a vitamin D insufficiency; and        -   administering a vitamin D insufficiency treatment to a            subject who is determined to have a vitamin D insufficiency.    -   XXIV. The method of paragraph XXIII, wherein the vitamin D is        selected from the group consisting of:        -   25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.    -   XXV. The method of any of paragraphs XXIII-XXIV, wherein the        determining of the level of free or bioavailable vitamin D        comprises use of a method selected from the group consisting of:        -   immunoassay; two-step immunoassay with antibody capture;            one-step immunoassay with immobilized antibody and            competitive detection; one-step immunoassay with immobilized            competitior and labeled antibody; fluorescence polarization            immunoassay; differential precipitation            (immunoprecipitation, affinity precipitation);            immunodepletion; and affinity binding chromatography;        -   and a method selected from the group consisting of:        -   radioimmunoassay; chemiluminescent immunosorbent assay;            electrochemiluminescent immunosorbent assay; fluorescent            immunosorbent assay; dye linked immunosorbent assay; liquid            chromatography tandem mass spectroscopy and high-pressure            liquid chromatography.    -   XXVI. The method of any of paragraphs XXIII-XXIV, wherein an        insufficiency of vitamin D indicates an increased risk of a        condition selected from the group consisting of:        -   decreased bone density; decreased bone mineral density; bone            fractures; bone resorption; rickets; osteitis fibrosa            cystica; fibrogenesis imperfect ossium; osteosclerosis;            osteoporosis; osteomalacia; elevated parathyroid hormone            levels; parathyroid gland hyperplasia; secondary            hyperparathyroidism; hypocalcemia; infection; cancer;            psoriasis; cardiovascular disease; renal osteodystrophy;            renal disease; end-stage renal disease; chronic kidney            disease; chronic kidney disease-associated mineral and bone            disorder; extraskeletal calcification; obesity; allergy,            asthama; multiple sclerosis; muscle weakness; rheumatoid            arthritis and diabetes.    -   XXVII. The method of any of paragraphs XXIII-XXVI, wherein the        treatment comprises administering a compound selected from the        group consisting of:        -   calcitriol; dihydrotachysterol; doxercalciferol;            paricalcitol; cholecalciferol and ergocalciferol.    -   XXVIII. An assay comprising:        -   analyzing a blood sample obtained from a subject to            determine a level of free vitamin D and albumin polypeptide;        -   wherein a level of bioavailable vitamin D is:            =(K _(alb) *[Alb]+1)*[Free Vitamin D]    -   XXIX. The assay of paragraph XXVIII, wherein a level of        bioavailable vitamin D lower than 25% of the mean value of        bioavailable vitamin D in a population of healthy subjects        indicates that the subject has a vitamin D insufficiency.    -   XXX. The assay of any of paragraphs XXVIII-XXIX, wherein the        vitamin D is selected from the group consisting of:        -   25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.    -   XXXI. The assay of any of paragraphs XXVIII-XXX, wherein the        determining of the level of albumin polypeptide comprises use of        a method selected from the group consisting of:        -   enzyme linked immunosorbent assay; chemiluminescent            immunosorbent assay; electrochemiluminescent immunosorbent            assay; fluorescent immunosorbent assay; dye linked            immunosorbent assay; immunoturbidimetric assay;            immunonephelometric assay; dye-based photometric assay;            western blot; immunoprecipitation; radioimmunological assay            (RIA); radioimmunometric assay; immunofluorescence assay and            mass spectroscopy.    -   XXXII. The assay of any of paragraphs XXVIII-XXXI, wherein the        determining of the level of free vitamin D comprises the use of        a method selected from the group consisting of:        -   immunoassay; two-step immunoassay with antibody capture;            one-step immunoassay with immobilized antibody and            competitive detection; one-step immunoassay with immobilized            competitior and labeled antibody; fluorescence polarization            immunoassay; differential precipitation            (immunoprecipitation, affinity precipitation);            immunodepletion; and affinity binding chromatography;        -   and a method selected from the group consisting of:        -   radioimmunoassay; chemiluminescent immunosorbent assay;            electrochemiluminescent immunosorbent assay; fluorescent            immunosorbent assay; liquid chromatography tandem mass            spectroscopy and high-pressure liquid chromatography.    -   XXXIII. The assay of any of paragraphs XXVIII-XXXII, wherein an        insufficiency of vitamin D indicates an increased risk of a        condition selected from the group consisting of:        -   decreased bone density; decreased bone mineral density; bone            fractures; bone resorption; rickets; osteitis fibrosa            cystica; fibrogenesis imperfect ossium; osteosclerosis;            osteoporosis; osteomalacia; elevated parathyroid hormone            levels; parathyroid gland hyperplasia; secondary            hyperparathyroidism; hypocalcemia; infection; cancer;            psoriasis; cardiovascular disease; renal osteodystrophy;            renal disease; end-stage renal disease; chronic kidney            disease; chronic kidney disease-associated mineral and bone            disorder; extraskeletal calcification; obesity; allergy,            asthama; multiple sclerosis; muscle weakness; rheumatoid            arthritis and diabetes.    -   XXXIV. The assay of any of paragraphs XXVIII-XXXIII, further        comprising the step of administering a vitamin D insufficiency        treatment to a subject who is determined to have a vitamin D        insufficiency.    -   XXXV. The assay of any of paragraphs XXVIII-XXXIV, wherein the        treatment comprises administering a compound selected from the        group consisting of:        -   calcitriol; dihydrotachysterol; doxercalciferol;            paricalcitol; cholecalciferol and ergocalciferol.

EXAMPLES Example 1 Bioavailable Vitamin D and Bone Mineral Density

Studies examining the relationship between total circulating25-hydroxyvitamin D (25(OH)D) levels and bone mineral density (BMD) haveyielded mixed results. Vitamin D binding protein (DBP), the majorcarrier protein for 25(OH)D, may alter the biologic activity ofcirculating vitamin D. Demonstrated herein is a test of the hypothesisthat free and bioavailable 25(OH)D, calculated from total 25(OH)D, DBPand serum albumin levels, is more strongly associated with BMD thanlevels of total 25(OH)D.

Total 25(OH)D, DBP, and serum albumin levels were measured in 49 healthyyoung adults enrolled in the Metabolic Abnormalities in College-AgedStudents (MACS) study. Lumbar spine BMD was measured in all subjectsusing dual X-ray absorptiometry. Clinical, diet, and laboratoryinformation was also gathered at this time. Free and bioavailable(free+albumin bound) 25(OH)D was determined and their associations withBMD were examined.

BMD was not associated with total 25(OH)D levels (r=0.172 p=0.236). Incontrast, free and bioavailable 25(OH)D levels were positivelycorrelated with BMD (r=0.413 p=0.003 for free, r=0.441 p=0.002 forbioavailable). Bioavailable 25(OH)D levels remained independentlyassociated with BMD in multivariate regression models adjusting for age,sex, body mass index, and race (p=0.03). Free and bioavailable 25(OH)Dare more strongly correlated with BMD than total 25(OH)D. These findingshave important implications for vitamin D supplementation in vitamin Ddeficient states.

Introduction

Vitamin D insufficiency is associated with decreased calcium absorptionand elevated levels of parathyroid hormone (PTH), (1) which may lead toexcessive bone resorption. (2) In some observational studies, higherlevels of 25-hydroxyvitamin D (25(OH)D) have been linked to increasedbone mineral density (BMD) and decreased risk of fracture. (3-7)Additionally, several randomized control trials suggest that vitamin Dsupplementation reduces the risk of fracture and increases BMD. (8-14)However, not all observational studies have confirmed the relationshipbetween 25-hydroxyvitamin D (25(OH)D) and BMD, especially in youngerpopulations or racial minorities. (15-19) Moreover, in severalrandomized trials, the effect of vitamin D supplementation on BMD orfracture risk has been modest, (8) absent, (20-22) or reversed. (23)

The free hormone hypothesis postulates that only hormones liberated frombinding proteins enter cells and produce biological action. (24) 25(OH)Dand 1,25-dihydroxyvitamin D (1,25(OH)2D) circulate bound to vitamin Dbinding protein (85-90%) and albumin (10-15%) with less than 1% ofcirculating hormone in its free form. (25) In mice, vitamin D bindingprotein (DBP) prolongs the serum half life of 25(OH)D and protectsagainst vitamin D insufficiency by serving as a vitamin D reservoir.(26) However, DBP also limits the biological activity of injected 1,2(OH)2D in mice(26) and inhibits the action of vitamin D on monocytes andkeratinocytes in vitro. (27-28). The significance of circulating DBPlevels with regards to vitamin D's biological action in humans isunclear.

The free fraction of 25(OH)D and the binding affinity constants for25(OH)D's interaction with DBP and albumin have previously beenmeasured. (29) Formulae for the calculation of free 25(OH)D levels basedon serum concentrations of total 25(OH)D, DBP, albumin, and have beendeveloped based on this data. Measured and calculated values of free25(OH)D are highly correlated. (29)

Materials and Methods

Subject Recruitment.

A cross-sectional study was conducted in a subset of healthy youngadults enrolled in the Metabolic Abnormalities in College Students study(MACS), a study designed to evaluate the prevalence of metabolicabnormalities in university students. (30) Subjects were healthy 18-31year old male and female students from private universities in theBoston area. 170 subjects were recruited through flyers postedthroughout the Massachusetts Institute of Technology (MIT) campus andthrough targeted emails to random members of the student population. Allsubjects provided written informed consent. The study was approved bythe MIT Committee on the Use of Humans as Experimental Subjects. 49subjects had sufficient sample for inclusion in this analysis and theircharacteristics are presented in Table 1.

Study Visit.

Subjects were instructed to fast for 12 hours prior to admission to theMIT Clinical Research Center as outpatients and underwent a baselineevaluation including a blood sample collection and various physiologicmeasurements. Structured interviews were conducted by study nurses tocollect standard clinical information, minutes of exercise per week (in30 minute increments), and medication/supplement use. Height wasmeasured using a standing stadiometer (Holton Ltd, Crymych, Dyfed, UK).Weight was measured using a calibrated scale (SECA, Hanover, Md., USA).Body mass index (BMI) was calculated as weight(kg)/[height(m)]².

Dietary Information.

Subjects completed a written food record 1 week prior to the day ofstudy, recording 4 full days of food intake, including one weekend day.During the study visit to the MIT CRC, a registered dietitian reviewedthe food record with the subject to clarify the quantities and sourcesof food consumed. Dietary intake data were then analyzed using NutritionData System for Research software version 2006/2007 (NutritionCoordinating Center, University of Minnesota, Minneapolis, Minn.).

Bone Density Measurement.

Subjects underwent total-body dual-energy x-ray absorptiometry (DEXA)(Hologic QDR-4500A; Hologic, Waltham, Mass., USA) to determine total andregional BMD. (31) Hologic phantoms were used to calibrate theinstrument. Lumbar spine BMD was used in this study as the measure ofBMD. Lumbar spine BMD is a preferred site for the diagnosis ofosteoporosis and the prediction of fracture. No hip BMD measurementswere available. (32-33)

Biochemical Analysis.

Baseline blood samples were frozen at −80° C. and stored for lateranalysis. 25(OH)D, serum calcium, albumin, and levels of PTH weremeasured in the Massachusetts General Hospital (MGH) clinicallaboratories. 25(OH)D2 and 25(OH)D3 levels were measured by liquidchromatography tandem mass spectrometry (LC-MS), with interassay CV's of9.1% for 25(OH)D₂ and 8.6% for 25(OH)D₃. Total 25(OH)D level wascalculated as the sum of 25(OH)D₂ level and 25(OH)D₃ level. Intact PTHwas measured by electrochemiluminescense immunoassay on the Cobas E160automated analyzer (Roche Diagnostics, Indianapolis, Ind.). InterassayCV for intact PTH measurement was 4.2%. Calcium and albumin levels weremeasured by dye-based photometric assays on an automated analyzer. DBPwas measured in duplicate by commercial enzyme linked immunosorbentassay (ELISA) (R&D Systems, Minneapolis, Minn., Catalog Number DVDBP0)according to the manufacturer's instructions. The assay was conductedafter diluting serum samples 1 to 2,000 in Calibrator Diluent RD6-11(R&D Systems Part Number 895489). Inter-assay CV was 8.5% at aconcentration of 40 ug/ml. The assay recovered between 93 and 110% of a100-200 ug/mL dose of exogenous vitamin D binding protein added to humanserum samples containing between 25-200 ug/mL of endogenous vitamin Dbinding protein. The manufacturer reports no significantcross-reactivity with human albumin, vitamin D3, or alpha-fetoprotein.In a subset of patients in whom adequate serum was available (N=45),total 1,25(OH)₂D was measured by LC-MS/MS in the Mayo Clinic MedicalLaboratories (Rochester, Minn., USA).

Calculation of Unbound 25(OH)D.

Free levels of 25(OH)D were calculated using two methods. Both methodsused the binding affinity constants between albumin and DBP and 25(OH)Dmeasured in a previous study which used centrifugal ultrafiltration todetermine the free fraction of 25(OH)D. (29)

Method 1:

Free levels of 25(OH)D were calculated using the following equation:

$\begin{matrix}{{{Free}{\mspace{14mu}\;}25({OH})D} = {\frac{{Total}\mspace{14mu} 25({OH})D}{1 + \left( {6 \times 10^{5} \times {Albumin}} \right) + \left( {7 \times 10^{8} \times D\; B\; P} \right)}.}} & (29)\end{matrix}$The reported correlation coefficient between calculated free 25(OH)Dusing this equation and measured free 25(OH)D by centrifugalultrafiltration is 0.925. (29) Free 1,25(OH)D levels were alsocalculated using this method. (25)

Method 2:

Free, bioavailable, and DBP-bound 25(OH)D were calculated usingequations described in Appendix 1 of this Example below. These methodsdefine bioavailable hormone as the fraction that is both free andalbumin-bound, i.e. the fraction not bound to circulating bindingproteins such as DBP.

Both calculation methods used the same affinity binding constants.Applied to the same measurements of total 25(OH)D, DBP, and albumin,they produce calculated free 25(OH)D values that are highly correlated(Spearman r=1), however the equations produce values that are an averageof 1.4% higher (data not shown). Because the equations also provide forseparate calculation of free, bioavailable, and DBP-bound 25(OH)D,Method 2 procedures were used for subsequent analyses of 25(OH)D levels.

Statistical Analysis.

Subject characteristics are reported as mean±SD unless otherwise noted.Non-normal variables including 25(OH)D levels, DBP levels, BMD, anddietary calcium intake levels showed skewed distributions and werenatural log transformed in order to meet the assumptions of parametricstatistical techniques. Exercise amount was dichotomized at 120 minutesper week. Pearson's correlation coefficients were calculated to assessthe relationships between 25(OH)D levels, BMD, and other continuousvariables. Independent samples t-tests were used to compare 25(OH)Dlevels, DBP levels, and BMD among subgroups defined by race, sex,exercise amount, and oral contraceptive use. Linear regression analysiswas used to test for the presence of an independent relationship between25(OH)D levels, DBP, and BMD after adjustment for factors previouslyreported to be associated with bone density including age, sex, BMI andrace. (5, 8, 12, 35-36) All analyses were conducted using STATAStatistical Software (College Station, Tex.) version 11. Two sidedp-values <0.05 were considered statistically significant.

Results

Subject characteristics are shown in Table 1. There was wide variationin levels of DBP, with concentrations ranging from 0.66 to 11.2 umol/L.Accordingly, calculated free and bioavailable 25(OH)D levels rangedwidely (Table 2). Total 25-hydroxyvitamin D levels were positivelycorrelated with DBP levels (r=0.335, p=0.019).

Total 25(OH)D levels were not correlated with BMD (r=0.172 p=0.236, FIG.1). Similarly, levels of DBP-bound 25(OH)D were not correlated with BMD(r=0.072, p=0.626). In contrast, free and bioavailable 25(OH)D levelswere both strongly correlated with BMD (r=0.413 p=0.003 for free andr=0.441 p=0.002 for bioavailable, FIG. 1). Bioavailable and free 25(OH)Dlevels were highly correlated with each other (r=0.985, p<0.001), butbioavailable 25(OH)D made up a larger portion of the total 25(OH)D withapproximately 350-fold higher concentrations of bioavailable 25(OH)Dcompared to free 25(OH)D. (Table 2) Total and calculated free levels of1,25(OH)₂D were not correlated with BMD (p>0.05). Total levels of1,25(OH)₂D were not associated with free or bioavailable 25(OH)D levels(p>0.05), nor was sex-adjusted alkaline phosphatase associated withtotal, free, or bioavailable 25(OH) D (p>0.05) Neither total norbioavailable 25(OH)D levels were correlated with serum calcium or PTHlevels (p>0.05). Of note, PTH levels fell between 15 and 51 ng/L (allwithin the normal range) and were not associated with BMD (r=−0.024,p=0.869).

Both total 25(OH)D and DBP levels were inversely associated with BMI(r=−0.300, p=0.036 for total and r=−0.542, p<0.001 for DBP).Bioavailable 25(OH)D was positively correlated with BMI (r=0.302,p=0.035). However, in this population, BMI was not correlated with BMD(r=0.160, p=0.271). Dietary calcium intake was correlated with total25(OH)D (r=0.339, p=0.021), but was not correlated with DBP,bioavailable 25(OH)D, or BMD (p>0.05). Levels of total and bioavailable25(OH)D, DBP and BMD among selected subgroups are shown in Table 3.Females had greater average total 25(OH)D levels than males, but averageDBP levels, bioavailable 25(OH)D levels and BMD did not differ betweenmales and females. Females reporting use of oral contraceptive pills(OCP) had higher average total 25(OH)D compared with females who did notreport OCP use, but average DBP and bioavailable 25(OH)D levels were notsignificantly different based on OCP use. Subjects with BMI greater thanor equal to 25 kg/m² (overweight subjects) had lower DBP levels thansubjects with BMI less than 25 kg/m² (normal weight subjects). Subjectswho reported exercising 120 minutes a week or more had higher averagetotal 25(OH)D levels than subjects who did not, but no significantdifference was found in average DBP levels, bioavailable 25(OH)D levels,and BMD. Average DBP levels in non-white subjects were lower than inwhite subjects (Table 3).

In multivariate models adjusting for age, sex, BMI and race,bioavailable 25(OH)D remained independently associated with BMD(p=0.03,Table 4). Bioavailable 25(OH)D was the only significant predictor of BMDin multivariate models. As the level of calculated bioavailable 25(OH)Dis dependent on the concentrations of total 25(OH)D, albumin, and DBP,it was separately assessed whether albumin or DBP was associated withBMD. DBP level was inversely correlated with BMD (r=−0.296, p=0.039)while serum albumin showed no association with BMD (r=0.156, p=0.285).In a multivariate linear regression model, total 25(OH)D became asignificant predictor of BMD only after adjustment for DBP level(B=0.089, p=0.040). Albumin was not associated with BMD in amultivariate model including DBP and total 25(OH)D (p=0.150).

Discussion

In light of conflicting reports concerning the relationship betweencirculating levels of 25(OH)D and BMD, serum levels of total 25(OH)D,DBP, and albumin were measured in a group of young healthy adults andassessed relationships between free 25(OH)D, bioavailable 25(OH)D, total25(OH)D and BMD. Without meaning to be limiting, the results describedherein are consistent with the free hormone hypothesis and suggest thatcirculating DBP is an inhibitor of the biological action of vitamin D inhumans. The similar associations between free and bioavailable vitaminlevels and BMD imply that, unlike binding to DBP, binding to albumindoes not inhibit the action of 25(OH)D. These results are consistentwith prior basic and clinical studies on DBP.

The results described herein support the hypothesis that DBP behavessimilarly to other serum hormone carrier proteins and have broadclinical applications. Like thyroid hormone binding globulin and sexhormone binding globulin, DBP may act as a serum carrier and reservoir,prolonging the circulating half-life of vitamin D, while at the sametime regulating its immediate bioavailability to target tissues. (24).In contrast to the megalin-mediated endocytosis described in renaltubular cells, our results imply that 25(OH)D gains access to sometarget cells by diffusion across cell membranes, similar to these othersteroid hormones. (24) Thus hormonal activity and sufficiency may bereflected by the amounts of bioavailable vitamin, not by total serumlevels. Currently, clinical testing for vitamin D insufficiency is basedupon measurement of total serum concentrations of 25(OH)D. (2) Yet, thedata described herein suggests that concentrations of total serumvitamin D may not be the best measure of vitamin D sufficiency. Forexample, patients with high levels of DBP may appear to be 25(OH)Dsufficient, but may actually be deficient in bioavailable vitamin.Conversely, in patients with low levels of DBP, total 25(OH)D will below, but these patients may actually have sufficient bioavailablevitamin. The maintenance of bioavailable 25(OH)D levels in obese andnon-white subjects, despite lower levels of total 25(OH)D raise thepossibility that variation in circulating DBP explains the apparentparadox of low 25(OH)D levels and higher BMD in black and overweightpatients seen in several previous studies, (5, 15-16, 35, 44-45).

The results described herein contrast with results of some prior studieslinking total 25(OH)D levels to BMD, but are consistent with otherstudies which failed to find such a relationship. (4-5, 15-18) In theseprior studies, DBP levels were not measured. Of note, total 25(OH)D andfree/bioavailable 25(OH)D levels are associated, and it is possible thata larger sample size would have enabled detection of a weak relationshipbetween total 25(OH)D and BMD. Prior studies that found thisrelationship generally had sample sizes greater than 200 and whencorrelation coefficients between 25(OH)D and BMD were reported, theywere less than 0.2. (4-5, 16, 19)

A relationship between 1,25(OH)₂D levels and BMD was not found. While1,25(OH)₂D is thought to be the active form of vitamin D, many tissuesexpress 1-α hydroxylase, and may be able to convert circulating 25(OH)Dto its active form locally. (46) Circulating 25(OH)D levels aregenerally considered to better reflect overall vitamin D stores. (2) Theresults describe herein are in agreement with this, suggesting thattotal circulating total or free 1,25(OH)₂D levels are not good measuresof vitamin D activity. This is analogous to the accepted model for themeasurement of thyroid hormone action, where free T4 levels are a bettermeasure of thyroid hormone action than circulating free T3 levels, eventhough T3 is the active form of the hormone. (47)

The use of standardized immunoassays for vitamin D, DBP, and albumincombined with standard calculation methods would allow the approachdescribed herein to be adopted with more confidence by other clinicallaboratories.

A wide distribution of DBP levels among our subjects and observed thatDBP was negatively associated with both high BMI and black race, both ofwhich have been associated with low 25(OH)D levels. Without wishing tobe limiting, one potential explanation is that 25(OH)D might itselfregulate the production of DBP. Lowering DBP levels would allow a higherfraction of DBP to be bioavailable in situations where total levels arelow. Other possible explanations for the observed associations betweenrace, BMI, and DBP levels include genetic factors and uptake ofcirculating DBP by adipose tissue.

Described herein is evidence that DBP modifies the relationship between25(OH)D and BMD in humans. Our data suggest that bioavailable 25(OH)Dlevels are a better of measure of vitamin D activity than total 25(OH)Dlevels, at minimum, with respect to bone metabolism. It is thereforepossible that by using total 25(OH)D levels as a measure of vitamin Dsufficiency, individuals may be misclassified as vitamin D sufficient orinsufficient. This may explain conflicting results of prior studies ofthe relationship between serum 25(OH)D concentrations and BMD.Determining which individuals have a true deficit in vitamin D may allowfuture vitamin D supplementation interventions to be targeted to thoseindividuals most likely to benefit. Additionally, use of bioavailable25(OH)D levels may further elucidate the nature of the relationshipbetween vitamin D and a wide range of outcomes including fracture, (7)infection, (49) cancer, (50) and cardiovascular disease. (51)

Appendix. Derivation of Calculated Free and Bioavailable25-Hydroxyvitamin D DEFINITIONS

-   -   D=25-hydroxyvitamin D (calcidiol), sum of both D2 and D3    -   Alb=albumin    -   DBP=Vitamin D binding protein, also known as Group-specific        component or Gc    -   [D_(Alb)]=concentration of albumin-bound vitamin D    -   [D_(DBP)]=concentration of DBP-bound vitamin D    -   [D]=concentration of free (unbound) D    -   [Total]=concentration of Total 25OH-D=[D_(DBP)]+[D_(Alb)]+[D]    -   [Bio]=concentration of Bioavailable D (Bioavailable=sum of free        and albumin-bound vitamin)=[D]+[D_(Alb)]    -   K_(alb)=affinity constant between vitamin D and albumin=6×10⁵        M⁻¹    -   K_(DBP)=affinity constant between vitamin D and DBP=0.7×10⁹ M⁻¹

Equations

Total 25(OH)-Vitamin D[Total]=concentration of 25(OH)-Vitamin D in g/mol÷400.5 g/moleGiven that [Total]=[D]+[D _(Alb) ]+[D _(DBP)]thus [D _(DBP)]=[Total]−[D _(Alb) ]−[D]  (Eq. 1)

Albumin[Alb]=serum albumin concentration in g/L÷66,430 g/mole[D]+[Alb]

[D _(Alb)]Albumin association constant K _(alb) =[D _(Alb]÷([D]·[Alb]))Thus [D _(Alb) ]=K _(alb) ·[Alb]·[D]  (Eq. 2)

-   -   (NB: [Alb] in this example denotes the concentration of free        non-vitamin bound albumin. However, given the low affinity        between albumin and Vit. D, the concentrations of total albumin        and unbound albumin are effectively equivalent ([Total        Albumin]≈[Alb]). As a result, [Alb] may be estimated accurately        by measurements of total serum albumin.)

DBP[Total DBP]=concentration of serum DBP in g/L 58,000 g/mole[DBP]=free unbound DBP and [D _(DBP)]=vitamin-bound DBPGiven that [D]+[DBP]

[D _(DBP)]And DBP association constant K _(DBP) =[D _(DBP)]÷([DBP]·[D])Thus [D]=[D _(DBP) ]÷K _(DBP) ÷[DBP]  (Eq. 3)Since [Total DBP]=sum of bound and unbound DBP=[DBP]+[D _(DBP)]Therefore [DBP]=[Total DBP]−[D _(DBP)]  (Eq. 4)

Solving for Free 25(OH)-Vitamin D

From Eqs. 3 and 4 we see that:[D]=[D _(DBP) ]÷K _(DBP)÷([Total DBP]−[D _(DBP)])  (Eq. 5)

If we substitute Eq. 1 into Eq. 2, we find that:[D _(DBP)]=[Total]−(K _(alb) ·[Alb]+1)·[D]  (Eq. 6)

Substituting Eq. 6 into Eq. 5 produces the following:[D]={[Total]−(K _(alb) ·[Alb]+1)·[D]}÷K _(DBP)÷([Total DBP]−{[Total]−(K_(alb) ·[Alb]+1)·[D]})

The equation is now limited to known constants (K_(DBP) and K_(alb)),measured values ([Total DBP], [Alb], and [Total]) and the dependentvariable for free vitamin D [D]. After propagating products and severalrearrangements we can further simplify this to fit the form of asecond-degree polynomial:ax ² +bx+c=0

Where x=[D]=the concentration of free 25(OH)-Vitamin D

a=K_(DBP)·K_(alb)·[Alb]+K_(DBP)

b=K_(DBP)·[Total DBP]−K_(DBP)·[Total]+K_(alb)·[Alb]+1

c=−[Total]

This polynomial may be solved for [D] using the quadratic equation:[D]=[−b+√b2−4ac]÷2a

-   -   After solving for free 25(OH)-vitamin D, we may then use Eq. 2        to calculate the concentration of bioavailable (non-DBP bound        vitamin):        [Bio]=[D]+[D _(Alb)]=(K _(alb) ·[Alb]+1)·[D]  (Eq. 7)

Example Calculation

Total 25(OH)-vitamin D=[Total]=40 ng/mL=1.0×10⁻⁷ mol/L

Total serum DBP=[Total DBP]=250 ug/mL=4.3×10⁻⁶ mol/L

Total serum albumin=[Alb]=4.3 g/dL=6.4×10⁻⁴ mol/L

K_(alb)=6×10⁵M⁻¹

K_(DBP)=7.0×10⁸M⁻¹

a=2.7×10¹¹

b=3325

c=−1×10⁻⁷

Calculated concentration of free 25(OH)D=3.01×10⁻¹¹ mol/L=12.1 pg/mL

Calculated concentration of bioavailable 25(OH)D=1.09×10⁻⁸ mol/L=4.6ng/mL

REFERENCES

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Example 2 Bioavailable Vitamin D and Mineral Metabolism

Prior studies have yielded conflicting results regarding the associationbetween 25-hydroxyvitamin D (25(OH)D) levels and mineral metabolism inend-stage renal disease (ESRD). Described herein are experiments testingthe hypothesis that bioavailable vitamin D, the vitamin D fraction notbound to vitamin D binding protein (DBP), would associate more stronglywith measures of mineral metabolism than total levels. Eighty ninepatients with previously measured 25(OH)D and 1,25-dihydroxyvitamin D(1,25(OH)₂D) levels were identified from a cohort of incident U.S.dialysis patients. Stored serum samples were used to measure DBP,determine bioavailable 25(OH)D and 1,25(OH)₂D using previously validatedformulae, and examine associations with measures of mineral metabolismand demographic factors. Both bioavailable 25(OH)D and bioavailable1,25(OH)₂D were correlated with serum calcium (r=0.26, p=0.01 andr=0.23, p=0.02, respectively) whereas this association was absent forboth total 25(OH)D (r=0.01, p=0.92) and total 1,25(OH)₂D (r=0.08,p=0.44). Racial differences in DBP and total 25(OH)D, but notbioavailable vitamin D, were observed. In univariate and multivariateregression analysis, only bioavailable 25(OH)D was associated withparathyroid hormone levels (p=0.007 and p=0.02, respectively).Accordingly, bioavailable 25(OH)D levels are better correlated withmeasures of mineral metabolism than total 25(OH)D levels in patients onhemodialysis.

Chronic kidney disease-associated mineral and bone disorder (CKD-MBD) isone of the most appreciated metabolic complications of CKD. Asindividuals progress toward end-stage renal disease (ESRD), decliningrenal 1a-hydroxylase activity leads to decreased conversion of25-hydroxyvitamin D (25(OH)D) to the active 1,25-dihydroxyvitamin D(1,25(OH)₂D). These metabolic changes are believed to precipitate thehypocalcemia and secondary hyperparathyroidism that characterizeCKD-MBD. Although 1,25(OH)₂D is thought to be the biologically activemoiety, the majority of vitamin D circulates as 25(OH)D. (1) Low levelsof 25(OH)D are common in ESRD; 79% of patients initiating dialysis have25(OH)D levels below 30 ng/ml, and serum levels below this threshold arenearly universal among black ESRD patients. (2)

The free hormone hypothesis suggests that protein-bound hormones arerelatively inactive while those liberated from binding proteins are freeto exert biological activity. (3) For some hormones (e.g. testosterone),binding to albumin is considerably weaker than to a specific bindingprotein. Thus, albumin-bound hormone is often grouped with the freefraction and referred to as the “bioavailable” fraction. The majority(85-90%) of circulating 25(OH)D and 1,25(OH)₂D is tightly bound tovitamin D binding protein (DBP), with a smaller amount (10-15%) looselybound to albumin. Less than 1% of circulating vitamin D exists in afree, unbound form. (4, 5) Described herein are experiments testing thehypothesis that the relationship between vitamin D and markers ofmineral metabolism (e.g. PTH and calcium) in ESRD would be strengthenedby use of DBP and albumin to determine bioavailable vitamin D levels.Given the patterns observed in other cohorts, it was furtherhypothesized that the lower 25(OH)D levels typically seen in blackdialysis patients would be associated with lower and not necessarilylower bioavailable vitamin D levels in this group. (6, 7)

Results

Baseline characteristics of the 94 subjects included in this analysis,which are similar to those of a typical US hemodialysis population, aresummarized in Table 5. None of the included subjects were recorded asreceiving treatment with activated vitamin D, ergocalciferol, orcholecalciferol before initiating dialysis.

Mineral Metabolism and Vitamin D.

Baseline corrected calcium levels, measured within 14 days of chronichemodialysis initiation, were not associated with total levels of either25(OH)D (r=0.01, P=0.92) or 1,25(OH)₂D (r=0.08, P=0.44). In contrast,calcium levels correlated positively with both bioavailable 25(OH)D(r=0.26, p=0.01) and bioavailable 1,25(OH)₂D (r=0.23, p=0.02). Theserelationships are plotted in FIG. 2. A single individual with thehighest bioavailable 25(OH)D and bioavailable 1,25(OH)₂D appeared to bean outlier with respect to the observed relationships, with both levelsover 4 standard deviations above the mean. To examine the impact of thissingle data point, a sensitivity analysis was performed by repeating theanalysis with this individual excluded. The relationship of calcium withbioavailable 25(OH)D (r=0.30, p=0.003) and bioavailable 1,25(OH)₂D(r=0.27, p=0.008) were both somewhat strengthened.

Phosphorus levels demonstrated no association with either total levelsof 25(OH)D (r=0.14, P=0.19) or 1,25(OH)₂D (r=−0.01, P=0.94). Similarly,neither bioavailable 25(OH)D (r=−0.10. P=0.32) nor bioavailable1,25(OH)₂D (r=−0.16, P=0.12) were significantly associated withphosphorus levels.

Alkaline phosphatase was not associated with either total orbioavailable forms of 25(OH)D or 1,25(OH)₂D (p>0.05 for allcomparisons).

The relationship between PTH and all four forms of vitamin D wereexamined in univariate and multivariate regression models. In univariatemodels, only bioavailable 25(OH)D was associated with PTH, with a −0.35log decrease in PTH for each log increase in bioavailable 25(OH)D(p=0.01). In a multivariate model controlling for age, gender, race, andsurvival status at one year, this relationship remained unchanged(β=−0.32, p=0.02). A third model adding calcium, phosphorus, andbioavailable 1,25(OH)2D levels demonstrated similar results (Table 7).As with the calcium findings, both the unadjusted and adjustedcoefficients became stronger when a single outlier was excluded(unadjusted: β=−0.40, p=0.003; adjusted: β=−0.36, p=0.01). In contrast,there was no significant association between total 25(OH)D and PTH (FIG.3).

Patient Factors and Vitamin D.

Older individuals had higher total 25(OH)D levels (r=0.31, P=0.003) andbioavailable 25(OH)D (r=0.21, p=0.04). Neither total nor bioavailable1,25(OH)₂D were associated with age. Female gender was associated withlower total 25(OH)D levels (median in men: 22.0 ng/dl, in women: 18.0ng/dl; p=0.03). While females had numerically lower median total1,25(OH)₂D and bioavailable 25(OH)D and 1,25(OH)₂D levels, none of thesedifferences were statistically significant.

Black individuals had lower total 25(OH)D levels (median: 15.2 vs 23.2ng/ml, p<0.001) but not bioavailable 25(OH)D levels (median: 3.8 vs. 2.8ng/ml, p=0.21). The contrast in racial differences between these twoforms of vitamin D was driven largely by lower DBP levels among blacks.This difference persisted even when examining only individuals whosurvived for one year on dialysis or those who died in this timeframe(Table 6). PTH levels did not differ significantly by race (median: 201pg/ml [black] vs. 168 pg/ml [white], p=0.47). Neither total norbioavailable 1,25(OH)₂D levels differed by race (p=0.07 and 0.49,respectively). Of note, we found no racial differences in systolic ordiastolic blood pressure, diabetes, or BMI.

The study was not specifically powered to address whether systolic anddiastolic blood pressure, BMI, and survival or a diagnosis of diabeticnephropathy or diabetes were associated with any form of vitamin D (datanot shown).

Sensitivity Analysis.

Sensitivity analyses were performed to address the possibility thaturemia might alter DBP's binding affinity with 25(OH)D or 1,25(OH)₂D.With DBP-binding coefficients that were 25% lower than those originallydetermined by Bikle, et al., (4, 5) bioavailable measures of both25(OH)D (r=0.26, p=0.01) and 1,25(OH)₂D (r=0.22, p=0.03) remainedassociated with corrected calcium. Similar results were observed with25% higher coefficients (bioavailable 25(OH)D:r=0.27, p=0.009;bioavailable 1,25(OH)₂D (r=0.24, p=0.02). Associations of bioavailable25(OH)D with PTH remained statistically significant in both cases, withassociation coefficients changing less than 12% in either univariate ormultivariate analyses.

Discussion

Using a retrospective cohort of incident dialysis patients, therelationship between measures of mineral metabolism (including serumcalcium and PTH) and both total and bioavailable levels of vitamin D wasexamined. Described herein are results indicating that bioavailable25(OH)D is associated with both corrected serum calcium levels and PTH,both of which are well-established measures of mineral metabolism inESRD, while total 25(OH)D demonstrates no such associations. This databuilds upon prior findings described elsewhere herein: analysis from twoseparate cohorts now support the hypothesis that bioavailable measuresof vitamin D, which take into account binding of vitamin D to albuminand DBP, are more relevant to biological outcomes than are total levels,which are currently the standard measure of vitamin D status.

Some in vitro studies suggest that DBP-binding limits vitamin D activityin multiple target cells. (8, 9) Studies of DBP-null mice have shownthat these animals display markedly reduced levels of 25(OH)D and1,25(OH)₂D compared with wild-type mice, with a markedly reducedhalf-life. (10) Despite their low vitamin concentrations, when thesemice are provided with a steady source of dietary vitamin D, they showno differences in serum calcium, phosphorus, alkaline phosphatase, andPTH compared to wild-type controls. These studies support theapplication of the free hormone hypothesis to vitamin D physiology, atleast for some biological actions. Despite these findings, uptake ofprotein-bound hormone in cells expressing megalin appears to beimportant for some processes, so the biology underlying the findingsdescribed herein may be more complex than is immediately apparent andwarrants further investigation. (11, 12)

Black patients were oversampled as the data presented in Example 1suggested blacks have lower DBP levels than whites, an observationsupported by the data described in this Example. As previously reported,the inventors and others observed that black race is associated withlower levels of total 25(OH)D. (2, 13) As might be expected from thesetwo parallel racial differences (lower total 25(OH)D and lower DBP inblacks vs. whites), the levels of bioavailable 25(OH)D are similar, asdescribed herein.

Despite similar bioavailable D levels between racial groups, andassociation between bioavailable 25(OH)D and PTH, black patients hadnumerically higher PTH levels than their white counterparts. Though thisdifference was not statistically significant in the sample used herein,larger samples from this cohort have found significantly higher levelsof PTH in black individuals. (14) Bioavailable 25(OH)D did not differ byrace, yet were negatively associated with PTH, suggesting that racialdifferences in PTH are not primarily driven by differences in 25(OH)D.Indeed, others have found that PTH levels in blacks are higher thanthose in whites, even in states of 25(OH)D sufficiency. (13)

Several studies have attempted to assess the metabolic consequences oflow 25(OH)D levels in advanced CKD and ESRD, but have yieldedconflicting results. Ergocalciferol, a form of nutritional vitamin Dthat can increase 25(OH)D levels, appears to affect parathyroid hormone(PTH) levels in stage 3, but not in stage 4, CKD. (15, 16) Moreover,some studies have demonstrated a significant association between 25(OH)Dlevels and PTH in ESRD, (17-19) while others have not. (20,21)Associations between 25(OH)D and serum calcium have been similarlymixed. (2, 17,22)

This contradictory data has led to confusion about the role thatrepleting 25(OH)D (e.g. with nutritional forms of vitamin D such ascholecalciferol or ergocalciferol) plays in the management of patientswith ERSD. (23,24) In order to study the role of vitamin D insufficiencyand identify patients who are most likely to benefit from repletion, itis critical to have a biologically relevant measure of vitamin D status.Notably, the experiments described herein failed to find any significantlink between total or bioavailable 1,25(OH)₂D and relevant measures ofmineral metabolism, echoing the general consensus that circulating serumlevels of the active hormone are not useful as a measure of vitamin Dstatus. (1)

A relationship between survival and vitamin D status was not found,though this sample had considerably less power to detect thisrelationship than prior studies, which have found that severe vitamin Dinsufficiency (typically defined levels <10 ng/ml) is associated withincreased mortality. (19,25,26)

None of the individuals in this analysis, who initiated dialysis in 2004or 2005, had been treated with activated vitamin D analogs prior toinitiating dialysis.

PTH is commonly used as a proxy for metabolic bone disease in dialysispatients, but has an imperfect association with bone disease. (27) Bonebiopsies and non-invasive measures of bone density and structure werenot available in this study and are potential targets for futureanalyses. As described in Example 1 herein, a relationship betweenbioavailable 25(OH)D and bone density in a healthy population, (6) hasbeen demonstrated but it is not certain that this relationship willextend to the ESRD population given known alterations in mineralmetabolism. Metabolic changes that accompany ESRD and/or dialysis, aswell as genetic variants in DBP or other relevant proteins, have thepotential to influence binding of 25(OH)D to DBP. Whereas thesensitivity analysis did not indicate that these factors are likely toaffect the fundamental findings of this study, studies that directlymeasure bound and unbound fractions could improve upon the initialestimates and the equations used herein. Lastly, it is possible thatmeasured 25(OH)D levels in this study were influenced by levels of24,25(OH)₂D. Confirmation of the findings described herein with assaysable to differentiate 25(OH)D, 24,25(OH)₂D, and 1,24,25(OH)₃D mayfurther elucidate these biological relationships.

This study provides additional evidence to support the notion thatbioavailable, rather than total, levels of vitamin D may be morerelevant measures of vitamin D status with respect to its actions onmineral metabolism. While mineral metabolism has been the traditionalfocus of vitamin D actions, recent data suggest that its actions may bemore widespread, with effects on the immune response, (28) hypertension,(29) and insulin sensitivity, (30), among others.

Methods

Accelerated Mortality on Renal Replacement (ArMORR) is a nationallyrepresentative prospective cohort study of incident chronic hemodialysispatients (n=10,044) who began renal replacement between Jul. 1, 2004 andJul. 30, 2005 at one of 1,056 dialysis centers in the U.S. operated byFresenius Medical Care, North America (FMC). (31) The ArMORR datasetcontains a broad range of demographic and clinical data includingco-existing medical conditions, laboratory results, as well as serum andplasma samples. Clinical data were collected prospectively, entereduniformly into a central database by practitioners at the point of care.All clinical data arriving at Fresenius undergo rigorous data qualityassurance and quality control (QA/QC) auditing. Blood samples collectedfor clinical care were shipped to and processed by a central laboratory(Spectra East, Rockland, N.J., USA). After processing for routineclinical testing, remnant samples were shipped on ice to the ArMORRInvestigators where the samples were aliquotted and stored in liquidnitrogen. This study was approved by the Institutional Review Board ofthe Massachusetts General Hospital, which waived the requirement forinformed consent, and conducted in accordance with its ethical standardsand the Declaration of Helsinki

Study Population.

Between Jul. 1, 2004 and Jun. 30, 2005, 10,044 incident hemodialysispatients were prospectively enrolled into ArMORR. Subjects wereidentified who had 25(OH)D and 1,25(OH)₂D levels previously measured aspart of a case-control survival study. (19) Based on prior results in ahealthy population, we set a minimum sample size of 80 subjects. Toensure adequate power for racial comparisons, an approximately equalnumber of black (n=24) and white (n=23) patients were randomly selectedfrom the controls, and an equal number of race-matched cases. Thus, thetotal sample size was n=94. Baseline laboratory values were measuredfrom samples collected within 14 days of dialysis initiation.

Assays.

Total 25(OH)D and 1,25(OH)₂D were previously measured from thawedsamples in duplicate using a commercially available radioimmunoassay(DiaSorin Inc, Stillwater, Minn., USA). The interassay coefficients ofvariation (CVs) for 25(OH)D were <3% at levels <30 ng/ml and for1,25(OH)₂D were <6.5% at levels <32.5 pg/ml. Intact PTH (1-84) wasmeasured using the Nichols Advantage Biointact-PTH assay by thecentralized laboratory.

DBP was measured in duplicate in thawed serum samples by commercialenzyme linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis,Minn., Catalog Number DVDBP0) according to the manufacturer'sinstructions. The assay was conducted after diluting serum samples 1 to2,000 in Calibrator Diluent RD6-11 (R&D Systems Part Number 895489).Inter-assay CV was 8.5% at a concentration of 40 μg/ml. The assayrecovered between 93 and 110% of a 100-200 μg/mL dose of exogenous DBPadded to human serum samples containing between 25-200 μg/mL ofendogenous DBP. There were no differences in the recovery of exogenousDBP in black patients or obese patients. The manufacturer reports nosignificant cross-reactivity with human albumin or vitamin D3. DBPlevels were below the detection limit in 5 black patients who diedwithin the first year of dialysis. These individuals were assigned a DBPvalue equal to the lowest detectable level (12.3 μg/d1).

Calculation of Bioavailable Vitamin D.

Equilibrium dialysis and centrifugal ultrafiltration dialysis havepreviously been used by some investigators to indirectly measure freevitamin D levels, allowing estimation of the binding affinity constantsfor 25(OH)D and 1,25(OH)₂D with DBP and albumin. (4, 5, 32) In thesestudies, calculated levels of free 25(OH)D and levels measured bycentrifugal ultrafiltration were highly correlated (r=0.925). (5)Bioavailable and free vitamin D were calculated as described in Example1 herein.

Bioavailable 1,25(OH)₂D levels were determined using the same approachusing affinity constants previously derived by centrifugalultrafiltration dialysis. (4) These affinity constants were previouslyvalidated in both healthy and cirrhotic individuals, (4, 5) but have notbeen directly assessed in hemodialysis patients. Therefore a sensitivityanalysis of the main findings was performed using DBP bindingcoefficients for 25(OH)D and 1,25(OH)₂D that were 25% higher or 25%lower than previously measured values.

Statistical Analysis.

Prior to analysis, given the role of albumin as a binding protein forboth vitamin D and calcium, serum calcium levels were corrected foralbumin using the following equation: corrected calcium=totalcalcium+0.8*(4-albumin). (34) Spearman correlation analysis wasperformed to assess linear associations. Group comparisons of vitamin Dlevels were performed using the Wilcoxon rank sum test. To examinemultivariable associations between bioavailable vitamin D and PTH, bothvariables (because of non-normal distribution) were natural-logtransformed and analyzed using multivariate linear regression. Allanalyses were conducted using STATA Statistical Software (CollegeStation, Tex.) version 11.

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J    Clin Endocrinol Metab. 1999 October; 84(10):3666-3672.-   34. Correcting the calcium. Br Med J. 1977 Mar. 5; 1(6061):598.

Example 3 Bioavailable Vitamin D in Pregnant Women

A pilot study in pregnant women enrolled in the MOMS cohort wasconducted to determine if VDBP modifies the relationship between firsttrimester 25(OH)D levels and subsequent development of preeclampsia orgestational diabetes. Although the sample sizes in pregnant women weresmall, total 25(OH)D levels were significantly lower among blacks (18.8[10.8-24.2] ng/mL, n=9), Hispanics (21.4 [16.7-27.7] ng/mL n=61), andAsians (22.5 [14.8-29.7] ng/ml, n=8) compared to white non-Hispanics(32.1 [26.3-36.6] ng/mL, n=127; P<0.03 for all comparisons). VDBP levelswere significantly lower in blacks (97.2 [73.6-361.2] μg/ml) compared towhites (504 [338-700] μg/ml, P<0.001). Bioavailable 25(OH)D levels weresimilar in white and black subjects.

Example 4 Bioavailable Vitamin D and Racial Variability

Black individuals consistently have low 25-hydroxy vitamin D (25[OH)D]levels¹⁵⁻¹⁹ and are at especially high risk for poor outcomes linked tovitamin D sufficiency.¹⁵ Paradoxically, blacks have higher BMD and alower risk of osteoporosis than whites.²⁰ Although 25(OH)D is the markercurrently considered most suitable for assessing vitamin Dstatus,^(2,21) studies have shown a weaker relationship of 25(OH)D toBMD in blacks and other minorities compared to whites.^(18,19,22,23)Importantly, the BMD-vitamin D relationship in blacks needsclarification,¹⁸⁻²⁰ and questions about how to define clinicallyrelevant vitamin D insufficiency, the impact of race on measures ofvitamin D status, how to reliably identify who needs supplementationwith vitamin D (and with how much) to favorably impact disease outcomesremain unanswered.^(2,15)

The research described herein has the potential to redefine who isvitamin D deficient and to significantly improve diagnosis and theefficiency of strategies for prophylaxis and treatment. The hypothesisthat invokes the free hormone hypothesis in regards to bioavailablevitamin D can be investigated using a large cohort of black and whitesubjects enrolled in the Healthy Aging in Neighborhoods of Diversityacross the Life Span (HANDLS) study. Studies described in Examples 1, 2and 3 herein in various populations (healthy volunteers, individualswith end-stage renal disease (ESRD), pregnant women) measured VDBP,total 25(OH)D and calculated free 25(OH)D. These results indicate that25(OH)D levels are directly correlated with VDBP levels and inverselycorrelated with free 25(OH)D levels. The data also reveal substantialracial differences in VDBP levels. Blacks had 25-60% lower VDBP levelsthan whites, while free and bioavailable 25(OH)D levels were similar. Inhealthy adults, non-VDBP bound 25(OH)D levels were strongly associatedwith BMD, whereas total 25(OH)D levels and PTH were not.²³ Afteradjusting for race in patients with ESRD, both VDBP and free 25(OH)D(but not total 25(OH)D), were associated with PTH.³¹ These correlations(or lack thereof) between unbound vs total 25(OH)D and BMD and racialdifferences in VDBP levels led to the hyposthesis that VDBP modifies therelationship between BMD and 25(OH)D.²³ Total 25(OH)D may not faithfullyreflect physiologically relevant vitamin D status, particularly inrelation to BMD and questions the clinical relevance of 25(OH)D assaysin all races.³³

VDBP levels may be the previously unrecognized link that explains theBMD-vitamin D paradox in blacks. Using stored blood samples from blackand white subjects enrolled in HANDLS (n˜2,200), the followinghypotheses can be tested: 1. Blacks have lower levels of VDBP and total25(OH)D levels, but similar levels of free and bioavailable 25(OH)D aswhites. 2. Free and bioavailable 25(OH)D levels are independentlyassociated with BMD in blacks and whites, inversely and linearlyassociated with PTH levels, and these associations are stronger thanthose between total 25(OH)D and BMD.

Aim 1: To determine VDBP, total and bioavailable 25(OH)D, and PTH levelsin blacks vs whites.

Aim 2: To test for associations between BMD, bioavailable 25(OH)D, andPTH compared to total 25(OH)D in blacks vs whites.

The studies described herein, (1) apply well-established mechanisms ofhormone biology; (2) demonstrate that bioavailable/free 25(OH)D is morestrongly associated with outcomes than total 25(OH)D; (3) can aid futureclinical trial designs in which vitamin D insufficiency will beredefined; (4) aim to individualize diagnosis and treatment based onrace to improve therapeutic and cost-efficiency of our limited healthcare resources, and (5) question current public health paradigms forclinical decision-making about who is vitamin D deficient, who should betreated, and who can avoid being treated.

25(OH)D Insufficiency is Even More Common in Blacks than in Whites.

According to current cutoffs defining vitamin D deficiency in terms of25(OH)D, approximately 50% of African Americans in the US are eitherchronically or seasonally at risk.⁴⁸ Racial disparities in vitamin Dstatus between blacks and whites may arise from insufficient dietaryintake or impaired conversion of vitamin D by sunlight due to skinpigmentation.¹⁶ Holick⁴⁸ reported that in Boston, 84% of black men andwomen >65 years old were vitamin D deficient at the end of the summer,which is typically when vitamin D levels are highest. Deficiency wasattributed to several factors including insufficient milk intake becauseof lactose intolerance, decreased synthesis of vitamin D3 in the skindue to pigmentation, and avoidance of sun exposure to minimize skinpigmentation.⁴⁸ According to the 1988-1994 National Health and NutritionExamination Survey (NHANES)III, 42% of black women (15-49 years old)were vitamin D deficient at the end of the winter compared to 4% ofwhite women.⁴⁹ Concentrations in serum of 25(OH)D measured duringdifferent seasons showed substantially lower levels in blacks (adjustedfor body weight and vitamin D intake) throughout all seasons and smallerseasonal increases during summer months than whites.⁵⁰ These racialdifferences are reinforced in more recent NHANES 2001-2006 data.^(22,51)Table 9 summarizes several representative studies reporting vitamin Dlevels in blacks vs whites. In most studies, the discrepancy betweenraces does not extend to 1,25 (OH)₂D levels, which are generally similarin blacks and whites.^(50,52)

As described in Example 2 herein, data from ArMORR (AcceleratedMortality in Renal Replacement which includes ESRD patients at theinitiation of dialysis prior to any vitamin D replacement) demonstratedthat mean 25(OH)D levels were 23.2±13.7 (SD) ng/mL in whites (n=653) and16.9±10.9 in blacks (n=372) (P<0.05). Similar results were found from arandomly-selected race-matched sample from ArMORR in which total 25(OH)Dwas 27.3±15.3 ng/mL in whites (n=23) and 16.4±10.1 in blacks(n=24)(P=0.004).³¹ Given that: (1) the incidence of vitamin Dinsufficiency is consistently higher in blacks than whites; (2) therange of total 25(OH)D cutoff levels used to define insufficiency iswide; and (3) correlation between 25(OH)D with mineral markers and BMDis lacking,^(23,40) the public health implications of continued relianceon total 25(OH)D for diagnosis and treatment are broad. The marker usedto diagnose and supplement vitamin D deficient states must beappropriate for racially diverse populations. Based on the datadescribed in Example 2, it is hypothesized that total 25(OH)D may not beuniformly applicable to all races. The definition of vitamin Dinsufficiency needs to be revisited.

Improving the understanding of vitamin D status among different races isexpected to have broadly significant therapeutic and public healthimplications. Specifically, by improving knowledge about vitamin Dbiology, the 10M's public health position on vitamin D replacement canbe refined to consider race and VDBP levels when determining vitamin Dtargets.

The inventors have previously reported a curvilinear relationshipbetween PTH and total 25(OH)D that PTH in older hospitalized patients.⁴⁰PTH was inversely correlated to total 25(OH)D levels <15 ng/mL. Asimilar strong inverse correlation was observed in a large (n=825)cohort of incident dialysis patients.⁴³ At higher total 25(OH)D levels(≧15 ng/mL), the correlation with PTH was not as clear.⁴⁰ As describedin Example 1, in younger healthy subjects whose mean total 25(OH)Dlevels were 25.7±11.1 ng/mL (64.2±27.7 nmol/L), PTH was not correlatedwith total, bioavailable 25(OH)D, or BMD. This suggests that theassociation between free or bioavailable 25(OH)D levels and BMD is notmediated via PTH in individuals whose vitamin D status is relativelynormal. However, due to the relatively small sample size of the healthycohort, these correlations may not have been evident. Example 2indicates that, after adjusting for race, free 25(OH)D (but not total25[OH]D) correlates with PTH.

The large HANDLS dataset (n˜2,200) can be used to determine if PTH isbetter and more linearly correlated with free- or bioavailable 25(OH)Dthan with total 25(OH)D. Additionally, the question of whether racialdifferences exist among these variables can be explored. By followingsubjects with BMD measurements at baseline for changes over time in theHANDLS cohort, the measures of vitamin D which best predict changes inBMD in different racial groups can be identified.

Why do blacks have lower levels of vitamin D yet higher BMD than otherraces? Despite having lower levels of 25(OH)D, blacks have higherBMD^(19,20) and a lower risk of osteoporotic fractures thanwhites.^(20,58,59) Although factors other than vitamin D are likely tocontribute, it has been hypothesized that blacks have adaptive responsesthat protect the skeleton even when 25(OH)D is low. 16 Skeletalresistance to parathyroid hormone (PTH) activity and bone-sparingadaptations that promote beneficial skeletal effects of active vitamin D(1,25 dihydroxyvitamin D [1,25(OH)₂D]) have been proposed to explainthis paradox.¹⁶ For example, moderately low 25(OH)D can inducePTH-stimulated synthesis of 1,25(OH)₂D in the kidney.^(60,61) Using datafrom NHANES 2003-2006, Gutierrez and colleagues²² observed that BMDdecreased (p<0.01) with serum 25(OH)D and calcium intake among whitesand Mexican-Americans, but not among blacks (p=0.2).²² They proposedthat relationships between 25(OH)D, BMD, and PTH differ by race. Otherstudies have also shown that the relationship between 25(OH)D and BMD inblacks is weaker than in whites or is nonexistent.^(18,19)

Higher PTH levels have been reported in blacks than innon-blacks.^(22,31) Lower PTH may be related to low 25(OH)D, anadaptation to minimize urinary calcium losses and increase 1,25(OH)₂Dactivity. However, racial differences in PTH level persist even whentotal 25(OH)D is high,²² and PTH suppression by 25(OH)D may occur at alower threshold in blacks versus non-blacks.^(22,62) Thus, in additionto racial differences between BMD and total 25(OH)D levels, therelationships between PTH and free- and bioavailable 25(OH)D vs total25(OH)D may also differ by race. It is proposed herein that the VDBPhypothesis will help explain or better understand these relationships.

Although the relationship of VDBP, 25(OH)D, and BMD has not been clearlyestablished in humans, animal studies suggest a role for VDBP inmodulating the rates of bioavailability, activation, and end-organresponsiveness of vitamin D metabolism,⁶⁶ as well as a role in theBMD-25(OH)D paradox in blacks. That is, despite having lower total25(OH)D levels, free and bioavailable 25(OH)D levels in blacks may infact be normal or even higher than normal, as a result of relatively lowVDBP concentrations as explained by the free-hormone hypothesis.Conversely, skin with light pigment captures vitamin D from theenvironment more readily, and higher VDBP levels may be an adaptationfor regulating bioavailable vitamin D. Genetic data are collected inHANDLS and phenotyping has been performed by the HANDLS investigatorswith results published in peer reviewed journals.^(67,68)

The significance of circulating VDBP levels with regard to thebiological activity of vitamin D in humans isunclear.^(2,18,19,21,22,24-26,30) Several properties that influence thebioavailability of vitamin D are analogous to those of the lipid-solubleandrogen hormone, testosterone (T). In the circulation, total T is 60%bound to sex hormone binding globulin (SHBG), whereas 38% isalbumin-bound and 2% is available as free-T.⁷⁵ Non-SHBG-bound T and freeT are the biologically active components of circulating T (bio-T).⁷⁶ Themethod used to calculate bio-T includes measured values of total T,albumin, SHBG and their binding constants into a mathematical model oftripartite binding. A strong correlation exists between measured bio-Tand calculated bio-T.⁷⁷ Using similar methods, concentrations of freeand bioavailable vitamin D may be calculated using measured affinityconstants to VDBP and albumin, as described in Example 1 and elsewhereherein. Calculated free D values are highly correlated with measuredvalues of free D, as validated by Bikle et al.^(38,70) and can be usedto estimate circulating free vitamin D concentrations.

Studies on the various circulating forms of vitamin D have shown thatvitamin D is 85-90% bound to VDBP, 10-15% is albumin-bound, and only 1%circulates freely.⁷⁰ Given that the majority of vitamin D is bound toVDBP, what is the role of VDBP in vitamin D physiology? Multiple animalmodels have demonstrated that VDBP is important as a high affinity serumreservoir for Vitamin D. VDBP-deficient animals have no high affinityserum carrier for the vitamin; as a consequence of this their serumvitamin concentrations are significantly decreased, and without a highaffinity carrier they rapidly excrete vitamin D in the urine. Togetherthese events cause mice to quickly develop a vitamin insufficiency whenput on diets low in Vitamin D.⁶⁶ Although these animals are prone torapidly develop vitamin insufficiency in the absence of dietaryvitamins, when dietary vitamin D is abundant, the animals are able tomaintain calcium homeostasis and do not appear to suffer fromhypovitaminosis.

In contrast, animals that express VDBP but are missing the receptorsrequired for renal resorption of VDBP from glomerular ultrafiltratedisplay an even more dramatic phenotype. A significant amount of VDBP(and albumin) are filtered by the glomerulus.^(72,78) The filtered VDBPand albumin are normally recovered in the proximal tubules, however, bymegalin/cubulin receptor-mediated endocytosis. In the absence ofmegalin, vitamin D is sequestered by VDBP to the urine, producing rapidand complete vitamin insufficiency even when provided with avitamin-enriched diet, and develop severe abnormalities in calciumhomeostasis and bone disease.⁷²

The conclusions drawn from these animal models support a model whereVDBP and its endocytic receptors act as serum reservoirs and provide amechanism for the prevention of urinary losses. Based upon tissueculture and animal model studies, it is unclear whether VDBP is involvedin the intracellular delivery of 25(OH)D to its target tissues. The highaffinity of extracellular VDBP for 25(OH)D may prevent spontaneousdissociation and diffusion into cells. Experiments in tissue culturehave shown that when vitamin D-responsive osteoblasts or monocytes aretreated with vitamin D, addition of VDBP in the media actually inhibits25(OH)D endocytosis and intracellular signaling.^(73,74) Furthermore,although VDBP-deficient mice have very low serum vitamin concentrations,if they are provided with sufficient vitamin D in their diet they do notsuffer from problems with calcium homeostasis, and they accumulatenormal amounts of 1,25(OH)₂D in their tissues.⁷³

Together, these biochemical, tissue culture, and animal model studiessuggest that although VDBP helps to prevent insensible urinary losses,retain serum vitamin levels, and maintain stable vitamin Dconcentrations between meals, it is not necessary for intracellulardelivery of 25(OH)D or its conversion to active 1,25(OH)₂D. If VDBP isnot absolutely necessary for intracellular delivery of 25(OH)D, andsince albumin-bound 25(OH)D is the second most abundant circulating formof the vitamin, an alternative pathway for vitamin D delivery to theproximal tubules of the kidney and other target tissues must beconsidered: endocytic delivery of albumin bound vitamin D. Albumin andVDBP are in the same protein family, and they both share megalin andcubulin as their endocytic receptors. The most important target forvitamin D delivery and its actions is the epithelium of the proximalconvoluted tubule (PCT). Recent evidence has unexpectedly emerged that alarge amount of albumin and VDBP are filtered through the glomerularbasement membrane and then resorbed in the proximal tubule.^(79,80) Inthe PCT, albumin is resorbed by cubulin-mediated endocytosis,⁷⁹ and VDBPis endocytosed by megalin binding (although cubilin may also play arole).^(72,78) Megalin and cubilin are part of the same receptorcomplex, and thus albumin and VDBP share very similar endocyticpathways. The fact that both these proteins (and any vitamin bound tothem) are delivered to the renal epithelium in such large amountssuggests that either one may be a vehicle of intracellular vitamindelivery. Once these proteins are inside, however, albumin may be theprincipal source of diffusible 25(OH)D given its low 25(OH)D affinitycompared to VDBP. It has recently been demonstrated that the largeamounts of endocytosed albumin are actually transported through therenal epithelium and back to the circulation intact by transcytosis.⁸¹Given the structural and evolutionary similarities between albumin andVDBP, and their shared endocytic receptors, it is hypothesized that VDBPalso participates in transcytosis.

The transcytosis of albumin (and possibly VDBP) through renal epithelialcells described above thus saves these cells from the burden of havingto degrade this mass in the lysosomes, which, based upon the estimatedamounts of albumin flux would be toxic to the cells. This recent findingis significant to vitamin D bioavailability in megalin-expressing targettissues because transcytosis would also provide for an ideal autocrinemechanism for efficient intracellular delivery of vitamin D that can beregulated. As albumin transits through the cells, it would release25(OH)D through spontaneous dissociation, providing a nearby source ofvitamin D to intracellular VDBPs, CYP27B1 hydroxylase, and VDRreceptors. In contrast, although VDBP should not release of much of itsbound vitamin during transit through the tubular epithelium, it wouldensure efficient recovery of 25(OH)D from the urine and its return tothe circulation.

Given this alternative model of vitamin D physiology, because themajority of total serum vitamin D is bound to VDBP, and becauseVDBP-bound vitamin represents an inert serum reservoir for vitaminstorage, although measurement of total 25(OH)D may indicate total bodystores, serum concentrations of total 25(OH)D will often not reflectvitamin bioactivity or sufficiency. Non-VDBP bound bioavailable vitaminD, on the other hand, may be a more faithful indicator of vitamin Dsufficiency. This model agrees with results from clinical studiesdescribed above herein and it provides a model that may explain thedifferences in 25(OH)D concentrations and differences in BMD betweenwhite and black subjects.

Comparing men with osteoporosis to men without osteoporosis, Al-oanziand colleagues²⁴ found that total 25(OH)D₃ levels were similar in bothgroups, but VDBP levels were significantly higher (P<0.001) Calculatedfree 25(OH)D3 and 1,25(OH)₂D3 were significantly lower in men with vswithout osteoporosis (p<0.00001).²⁴ Whereas total 25(OH)D3 levelsprovided only a crude estimate of vitamin D status, measurement of freehormones provided more biologically relevant information. As describedherein in Example 1 (young healthy adults; n=49) BMD is notwell-correlated with total 25(OH)D but that free and bioavailable25(OH)D are much more strongly associated with BMD. Despite widevariation in VDBP concentrations in the study cohort, mean VDBP levelswere significantly lower in nonwhite than in white subjects (2.87±2.04,4.94±2.43, respectively; P<0.001). As demonstrated in Example 2 herein,a randomly selected group of racially-matched ESRD patients suggestedthat VDBP may also mediate vitamin D activity in ESRD. Lower VDBP levelswere found in blacks vs whites. Serum calcium correlated with free25(OH)D and 1,25(OH)₂D, but not with total 25[OH]D and free 25(OH)D andVDBP levels—but not total 25[OH]D—were significantly associated withPTH. As discussed in Example 3 herein, a study in pregnant women alsorevealed lower VDBP and total 25(OH)D levels in blacks vs whites,whereas bioavailable 25(OH)D was similar regardless of race.

On the basis of the data described herein, it is hypothesized that VDBPmodifies the relationship of BMD and 25(OH)D. More specifically,decreased concentrations of VDBP in nonwhites may explain the loweraverage concentrations of total 25(OH)D that have been consistentlyreported herein and elsewhere. Because of lower VDBP levels,bioavailable vitamin D will be normal or even increased, perhapsexplaining the apparent reduced risk of osteoporosis in blacks comparedto whites.

The invention described herein incorporates innovative hypotheses andapproaches that: 1. Apply mechanisms of hormone biology that arewell-established in other steroid hormone research (e.g. testosteroneand thyroid hormones) to explain the biological actions of vitamin D; 2.solves a complex problem that has puzzled clinicians for decades; 3. Islikely to impact the design of future clinical trials in which vitamin Dinsufficiency is redefined by decreased bioavailable vitamin D levels;4. Advances the applicability of the 25[OH]D assay that has long beenconsidered the gold-standard for determining vitamin D status andproposes a novel alternative (free/bioavailable D) that may solveperplexing inconsistencies in outcomes in different races that may besecondary to vitamin D status; 5, Advances a contemporary principle thatdiagnosis and treatment should be individualized based on, at least raceand gender, to improve the therapeutic and cost-efficiency of limitedhealth care resources.

Test Hypotheses in HANDLS Subjects.

The hypotheses described below can be explored (FIG. 5) by measuringtotal 25(OH)D and VDBP levels in stored serum samples from black andwhite subjects with baseline BMD measurements enrolled in HANDLS:33 1.Blacks have lower levels of VDBP and total 25(OH)D levels, but similarlevels of free and bioavailable25(OH)D as whites. 2. Free andbioavailable 25(OH)D levels are independently associated with BMD inblacks and whites, and are inversely and linearly associated with PTHlevels, and the associations are stronger than those between total25(OH)D and BMD.

Levels of free and bioavailable vitamin D can be calculated from total25(OH)D, VDBP and serum albumin measurements as shown below.^(38,70,76)Data will be examined using multivariate analyses for the presence ofassociations between BMD and free- and bioavailable 25(OH)D, and forassociations between free- and bioavailable 25(OH)D and PTH in black vswhite subjects. Adjustments for covariates such as: history ofosteoporosis, age, sex, pre-menopausal status, smoking and alcohol use,oral vitamin D and calcium intake, use of bisphosphonates, exercise, andbody mass index can be made.

The HANDLS Study.

The HANDLS study is being conducted as part of the National Institutionof Aging Intramural Research Program (NIAIRP). Planned as a 20-yearlongitudinal study, HANDLS is designed to test hypotheses about agingand health disparities in minority and poor populations by evaluatingdifferences in rates and risks for pathological conditions associatedwith aging within diverse racial, ethnic, and economic groups. Data onphysical, genetic, biologic, demographic, psychosocial, andpsychophysiological parameters from a fixed cohort of 3,722 black andwhite participants in higher and lower socioeconomic status are beingcollected.

Population being Studied.

HANDLS participants are a fixed cohort of community-dwelling black andwhite adults aged 30-64. Participants in HANDLS have been recruited from12 pre-determined neighborhoods (groups of contiguous census tracts)comprising the geographic area of Baltimore city and South Baltimore.The population comprises a 4-way factorial cross of age (seven 5-yearage bands between 30-64), sex, race, and socioeconomic status indexed bypoverty status.

Variables Available.

The HANDLS database contains information obtained from householdinterviews of black and white participants about their health status,health service utilization, psychosocial factors, nutrition,neighborhood characteristics, and demographics. In addition, mobilemedical research vehicles deployed every three years collect data (over˜20 years) from the same participants on: medical history and physicalexamination, dietary recall, cognitive evaluation, psychophysiologyassessments including heart rate variability, arterial thickness,carotid ultrasonography, assessments of muscle strength and bonedensity, and laboratory measurements (blood chemistries, hematology,biomarkers of oxidative stress and biomaterials for genetic studies).

The serum levels of total 25(OH)D, VDBP, albumin, PTH can be examinedand bioavailable 25(OH)D calculated in all HANDLS subjects with baselineBMD measurements (˜2200). Associations between VDBP levels, total25(OH)D, and PTH levels can be tested for. In addition to baselineassessments, 5 follow-up triennial assessments are being collected aspart of HANDLS over ˜20 years. At least one follow-up measure at Year 3(and as many as possible for the duration of the funding period) for anexploratory evaluation of BMD and related outcomes (ie, osteoporosis,fractures).

Blood Available.

Approximately 2,200 participants of HANDLS have baseline BMDmeasurements and stored serum samples. Followup BMD data, and bloodsamples collected at Year 3 (and possibly Year 6 as funding allows) canbe obtained to evaluate associations between BMD and related outcomesand total, bioavailable, and free 25(OH)D over time.

Analysis Techniques.

Stored serum samples from the HANDLS repository collected at baselineand at Years 3 and 6 (as feasible) can be used. Total 25(OH)D levels canbe measured by high performance liquid chromatography/massspectrophotometry. Albumin can be measured on standard automatedplatforms. VDBP can be measured using a commercially available ELISA(R&D Systems) as described in Example 1 herein. Interassay CVs andintra-assay CVs for this assay are 5.7% and 7.4% respectively.

In order to validate the calculated estimates of free 25(OH)D and testwhether these methods are valid in both black and white subjects, free25(OH)D can be measured directly in a subset of samples (n=200) asdescribed previously.⁸² Since blood sampling dates are captured, seasoncan be included as a covariate in the modeling of vitamin Dmeasurements.

The PTH assay can be performed using an electrochemiluminescenceimmunoassay “ECLIA” on the Cobas E601 analyzer (Roche diagnostics). Theassay for determining intact PTH employs a sandwich test principle inwhich a biotinylated monoclonal antibody reacts with the N-terminalfragment (1-37) and a monoclonal antibody labeled with a rutheniumcomplex reacts with the C-terminal fragment (38-84). The test canperformed using 300 μL of EDTA plasma, which provides more stable testresults than serum.

Power Calculations.

To determine VDBP, total and bioavailable 25(OH)D, and PTH levels inblack vs white subjects. Power was calculated assuming a 2-sided t-testwith α=0.05 for differences in black vs white subjects for total 25(OH)D(primary variable of interest). Although no previous studies haveexplored this measure among different racial groups, bioavailable25(OH)D is derived from total 25(OH)D and is a secondary variable ofinterest and it can constitute a reliable proxy. The weighted means andstandard deviations were derived from 5 published reports^(18,19,83-85)constituting a total of 14,402 whites and 7,790 blacks with clinical anddemographic characteristics similar to what we expect from the HANDLSpopulation (relatively healthy population, predominantly middle-aged).From these studies, an expected mean and standard deviation were derivedfor total 25(OH)D among whites and blacks (28.07±3.71 ng/mL and17.88±12.28 ng/mL, respectively). The sample size of nearly 2,200 HANDLSsubjects can be expected to be adequate to detect primary and secondaryvariables of interest with a power of 0.8, even with the possibility ofinvalid measurements and missing data (estimates at <5% of availableobservations).

To Test for Associations Between BMD, Bioavailable 25(OH)D, and PTHCompared to Total 25(OH)D in Black Vs White Subjects.

To obtain estimates for whole body BMD, a random sample of 40 subjects,stratified by race, was selected from all available HANDLS subjects withnon-missing whole body BMD and total 25(OH)D measurements. Power wascalculated assuming a 2-sided p-value with o=0.05. Based on preliminaryanalysis, r=0.29 between total 25(OH)D and whole body BMD in whites andr=0.24 among blacks was obtained. Assuming a correlation betweenbioavailable 25(OH)D and whole body BMD to be ˜30% higher, based onfindings from the MACS study, 23 the linear relation between thesevariables can be approximated to be r=0.38 among whites and r=0.31 amongblacks. Based on these estimates, the reduced R² among whites and blacks(R²=0.14 and R2=0.10, respectively) for multiple linear regressionanalyses was calculated. The sample size of nearly 2,200 HANDLS subjectsis adequate to detect the primary and secondary variables of interestwith a power of 0.8, even with the possibility of invalid measurementsand missing data (estimates at <5% of available observations).

Statistical Analysis

Preliminary examination of VDBP, total, bioavailable 25(OH)D, and PTHlevels include assessment of the plausibility of values through observedranges. Measures of central tendency and dispersion can be documented.As initial examination of VDBP, total, bioavailable 25(OH)D, and PTHlevels have shown highly, right-skewed distributions, these variablescan be assessed for normality through use of visualization techniquessuch as quantile-quantile plots and testing by Shapiro-Wilkes tests bothfor the overall sample and stratified by race. If VDBP, total,bioavailable 25(OH)D, and PTH levels are found to be normallydistributed, mean levels between black and white subjects can becompared through the use of two-samples t-tests. Otherwise, thedistributions between black and white subjects will be compared throughthe use of nonparametric statistics such as Wilcoxon rank sum tests.Two-tailed p-values of <0.05 will be considered statisticallysignificant.

BMD can be preliminarily be examined for plausibility of values andmeasures of central tendency and dispersion will be documented. Thedistribution of BMD can be inspected both through visualization andthrough formal statistical testing. Exploratory data analysis can beperformed through the use of scatterplots to inspect the relationshipbetween BMD, total 25(OH)D, bioavailable 25(OH)D, and PTH. If a linearrelationship is found between variables, Pearson Product-MomentCorrelation Coefficient or Spearman's Rank Correlation Coefficient (inthe case of outliers) can be used to examine associations between BMD,total 25(OH)D, bioavailable 25(OH)D, and PTH. Based on the strength ofthe relationships found between bioavailable 25(OH)D, total 25(OH)D, andPTH and BMD, multivariable linear regression models can be constructedto determine the independent association of bioavailable 25(OH)D, total25(OH)D, and PTH to BMD adjusting for other covariates, includinghistory of osteoporosis, age, sex, pre-menopausal status, smoking andalcohol use, oral vitamin D and calcium intake, use of bisphosphonates,exercise, and body mass index. Seasonality can be explored in all modelsby controlling for month of data collection and if seasonality is found,a cyclic regression curve can be included in the models.

The relationships between covariates and BMD, vitamin D levels, and PTHcan be explored through the use of independent samples t-tests (forbinary covariates) and simple linear regression (for continuouscovariates) as well as through visualization techniques includingboxplots and scatterplot matrices. Predictive variables can be selectedthrough the use of Least Angle Regression (LAR)⁸⁶ or Least AbsoluteShrinkage and Selection Operator (LASSO)⁸⁷ since both have proved to bemore reliable than the traditional stepwise variable selection method.Covariates central to our hypotheses or that have been shown in previousliterature to be clinically relevant to 25(OH)D, total 25(OH)D, and PTHand BMD can be left in the models irrespective of selection. Modelassessment can be performed through a variety of procedures.Collinearity can be assessed through correlations between covariates andthe variance inflation factor (VIF). Residuals can be examined throughstandardized residual plots and normality can be tested by Shapiro-Wilktests. Component plus residual plots will provide a means to investigatethe linearity between continuous predictors with BMD controlling for theeffects of other predictors in the model. The degree of overlap betweenLOESS curves and the regression line can be used to assess linearity ofthe predictors. Diagnostics of influential data points can be identifiedthrough Cook's distances (D) where observations with (Di>4/n) will besuspected of influence. A jack-knife approach can be used to comparemodels with and without influential observations and the percentage oferror attributed to individual observations can be documented.Observations with high Cook's distances but biologically plausiblevalues can be considered for inclusion in the final models. The finalcandidates of models can be compared for Akaike Information Criterion(AIC) value as well as adjusted Rsquared value.

Inclusion and formal testing of interactions between all covariates,with special attention paid to the interaction between race andsocioeconomic status, can be incorporated into un-stratifiedmultivariable models. Nonsignificant interaction terms can be droppedfrom final models. Significant interaction terms can be visuallyinspected through the use of Trellis graphs and the associations betweencovariates and BMD can be interpreted according to the level of theinteractive predictor. Multivariable models cam be conducted stratifiedby race and will be analyzed in a similar manner with the exclusion ofinteraction terms including race.

As CVD has been linked to 25(OH)D status, exploratory analyses can beperformed using available data to detect any relationships betweenbioavailable, total 25(OH)D, and PTH and carotid intimal medialthickness and blood pressure at baseline.

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Braunstein G D. In: Basic & Clinical Endocrinology. 5th ed.    Stamford, Conn.: Appleton & Lange; 1997:422-452.-   76. Vermeulen A, Verdonck L, Kaufman J M. A critical evaluation of    simple methods for the estimation of free testosterone in serum. J    Clin Endocrinol Metab 1999; 84:3666-72.-   77. Emadi-Konjin P, Bain J, Bromberg I L. Evaluation of an algorithm    for calculation of serum “bioavailable” testosterone (BAT). Clin    Biochem 2003; 36:591-6.-   78. Nykjaer A, Fyfe J C, Kozyraki R, et al. Cubilin dysfunction    causes abnormal metabolism of the steroid hormone 25(OH) vitamin    D(3). Proc Natl Acad Sci USA 2001; 98:13895-900.-   79. Amsellem S, Gburek J, Hamard G, et al. Cubilin is essential for    albumin reabsorption in the renal proximal tubule. J Am Soc Nephrol    2010; 21:1859-67.-   80. Russo L M, Sandoval R M, McKee M, et al. The normal kidney    filters nephrotic levels of albumin retrieved by proximal tubule    cells: retrieval is disrupted in nephrotic states. Kidney Int 2007;    71:504-13.-   81. Russo D, Corrao S, Miranda I, et al. Progression of coronary    artery calcification in predialysis patients. Am J Nephrol 2007;    27:152-8.-   82. van Hoof H J, Swinkels L M, Ross H A, Sweep C G, Benraad T J.    Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by    symmetric (rate) dialysis. Anal Biochem 1998; 258:176-83.-   83. Gutierrez O M, Farwell W R, Kermah D, Taylor E N. Racial    differences in the relationship between vitamin D, bone mineral    density, and parathyroid hormone in the National Health and    Nutrition Examination Survey. Osteoporos Int 2011; 22:1745-53.-   84. Reis J P, Michos E D, von Muhlen D, Miller E R, 3rd. Differences    in vitamin D status as a possible contributor to the racial    disparity in peripheral arterial disease. Am J Clin Nutr 2008;    88:1469-77.-   85. Cauley J A, Danielson M E, Boudreau R, et al. 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TABLE 1 Characteristics of the Study Population (n = 49). Data arepresented as n (%) for categorical variables and mean ± standarddeviation for continuous variables. Mean ± SD n (%) Age (years) 23.5 ±3.4  Body Mass Index (kg/m²) 22.43 ± 2.96  Sex Male 27 (55.1%) Female 22(44.9%) Race White 31 (63.3%) Non-White 18 (36.7%) Exercise Amount >120minutes per week 21 (42.9%) ≦120 minutes per week 26 (53.1%) Unknown  2(4.1%) Vitamin D Binding Protein (umol/L) 4.19 ± 2.49 Albumin (g/L)42.47 ± 3.94  Serum Calcium (mmol/L) 2.30 ± 0.19 Parathyroid Hormone(ng/L) 29.86 ± 8.25  Dietary Calcium Intake (mg/day) 925.85 ± 421.76Lumbar Spine Bone Mineral Density (g/cm²) 1.05 ± 0.14

TABLE 2 Serum Levels of Vitamin D. Total 25(OH)D levels were measuredalong with albumin and vitamin D binding protein (DBP). DBP-bound,albumin-bound, free, and bioavailable 25(OH)D (free and albumin bound)were calculated Mean ± SD Total 25(OH)D (nmol/L) 64.23 ± 27.70 DBP-Bound25(OH)D (nmol/L) 54.66 ± 26.32 Albumin-Bound 25(OH)D (nmol/L) 9.55 ±6.72 Free 25(OH)D (pmol/L) 25.37 ± 18.52 Bioavailable 25(OH)D (nmol/L)9.58 ± 6.74

TABLE 3 Bone Mineral Density, DBP Levels, and 25(OH)D Levels in SelectSubgroups. Values are reported as mean ± standard deviation. DBP =vitamin D binding protein. BMD = bone mineral density. OCP = oralcontraceptive pill. Groups were compared using t-tests after natural logtransformation of total 25(OH)D, DBP, bioavailable 25(OH)D levels andBMD. Total 25(OH)D Total DBP Bioavailable 25(OH)D L-Spine BMD n (nmol/L)(umol/L) (nmol/L) (g/cm²) Sex Male 27 52.79 ± 19.31 3.90 ± 2.09 8.36 ±5.36 1.04 ± 0.14 Female 22 78.28 ± 30.28 4.53 ± 2.92 11.08 ± 8.01  1.07± 0.13 p-value <0.001 0.493 0.113 0.371 OCP Use (Females Only) Yes 7107.33 ± 27.87  6.27 ± 3.76 12.83 ± 11.64 1.06 ± 0.21 No 15 64.73 ±20.59 3.71 ± 2.12 10.26 ± 5.98  1.07 ± 0.08 p-value <0.001 0.152 0.8410.646 Body Mass Index <25 kg/m² 39 66.0 ± 26.9 4.63 ± 2.49 8.59 ± 5.281.04 ± 0.12 ≧25 kg/m² 10 57.2 ± 25.0 2.42 ± 1.61 13.43 ± 10.19 1.09 ±0.19 p-value 0.284 0.003 0.073 0.438 Exercise ≧120 Minutes/Week 21 73.33± 26.11 4.14 ± 2.56 11.76 ± 8.35  1.09 ± 0.13 <120 Minutes/Week 26 58.66± 27.97 4.20 ± 2.58 8.19 ± 4.85 1.03 ± 0.13 p-value 0.038 0.863 0.0890.165 Race White 31 68.84 ± 28.65 4.94 ± 2.43 7.84 ± 3.92 1.03 ± 0.10Non-White 18 56.30 ± 24.76 2.87 ± 2.04 12.56 ± 9.29  1.08 ± 0.18 p-value0.138 <0.001 0.065 0.346

TABLE 4 Bioavailable 25(OH)D Predicts Bone Mineral Density (BMD).Bioavailable 25(OH)D and BMD were natural log transformed prior toanalysis. The coefficient (B) represents the average unit increase in lnBMD for each unit increase in ln bioavailable 25(OH)D. P-value is thestatistical significance of the relationship between bioavailable25(OH)D and BMD after controlling for potential confounders. Thus, therelationship between bioavailable 25(OH)D and BMD remains significantafter adjusting for potential confounders. Model B P-value Adjusted R²Bioavailable 25(OH)D 0.092 0.002 0.177 Bioavailable 25(OH)D, 0.072 0.0290.180 age, sex, race, and BMI

TABLE 5 Characteristics of the population (n = 94) Median (IQR) or n (%)Age, years 65 (50-74) Male 55 (59%) Black race 48 (51%) Survived atleast one year on dialysis 47 (50%) Body mass index 25 (22-30) Systolicblood pressure, mm Hg 140 (123-153) Diastolic blood pressure, mm Hg 73(61-81) Total 25(OH)D, ng/ml 20 (13-28) Total 1.25(OH)₂D, ng/ml 9.5(5-16) Parathyroid hormone, pg/ml 190 (96-307) Corrected Calcium, mg/dl8.9 (8.5-9.4) Phosphorus, mg/dl 4.2 (3-5.5) Alkaline phosphatase, mg/dl82 (66-112.5) Albumin, g/dl 3.4 (3.0-3.8) Vitamin D binding protein,μg/ml 158 (69-217) Bioavailable 25(OH)D, ng/ml 3.4 (2.2-5.0)Bioavailable 1.25(OH)₂D, pg/ml 2.2 (1.1-3.8)

TABLE 6 Race and vitamin D levels. Black individuals had lower total,but not bioavailable, 25(OH)D levels when compared with their whitecounterparts. Survivors are patients who survived for at least one yearafter initiating hemodialysis, while non-survivors died within thisyear. All values represent group medians. Blacks Whites p Total 25(OH)D(ng/ml) 15.1 23.1 <0.001 Bioavailable 25(OH)D (ng/ml) 3.8 2.8 0.21 Total1.25(OH)₂D (pg/ml) 8 11.5 0.07 Bioavailable 1.25(OH)₂D (pg/ml) 2.2 2.20.48 DBP (μg/ml) 75 189 <0.001 Survivors: DBP (μg/ml) 88 195 0.004Non-survivors: DBP (μg/ml) 58 183 <0.001 PTH (pg/ml) 201 168 0.47

TABLE 7 PTH and bioavailable 25(OH) vitamin D. In univariate andmultivariate analyses, bioavailable 25(OH) vitamin D levels wereconsistently associated with PTH (corresponding p values displayed). PTHand bioavailable vitamin D levels were log transformed prior toanalysis, thus β = −0.36 suggests that a 25% increase in bioavailable25(OH)D is associated with 7.7% decrease in PTH ((1.25^(−0.36) − 1)*100= −7.7). β p Bioavailable 25(OH)D alone −0.36 0.007 Multivariate modeladding age, gender, race −0.33 0.02 Multivariate model with abovevariables plus survival −0.32 0.02 status at 1 year Multivariate modelwith above variables plus calcium, −0.39 0.02 phosphorus, bioavailable1.25(OH)₂D

TABLE 8 Calculation of bioavailable vitamin D levels Number Equation 1[D_(DBP)] = [Total Vitamin D] − [D_(Alb)] − [D_(free)] 2 [D_(Alb)] =K_(alb) · [Alb] · [D_(free)] 3 [D_(free)] = [D_(DBP)] ÷ K_(DBP) ÷[DBP_(free)] 4 [DBP_(free)] = [Total DBP] − [D_(DBP)] From equations 3and 4 5 [D_(free)] = [D_(DBP)] ÷ K_(DBP) ÷ ([Total DBP] − [D_(DBP)])From equations 1 and 2 6 [D_(DBP)] = [Total Vitamin D] − (K_(alb) ·[Alb] + 1) · [D_(free)] 7 [D_(free)] = {[Total Vitamin D] − (K_(alb) ·[Alb] + 1) · [D_(free)]} ÷ K_(DBP) ÷ ([Total DBP] − {[Total Vitamin D] −(K_(alb) · [Alb] + 1) · [D_(free)]}) This can be simplified to fit asecond-degree polynomial (ax² + bx + c = 0) where x = [D_(free)]: a =K_(DBP) · K_(alb) · [Alb] + K_(DBP) b = K_(DBP) · [Total DBP] − K_(DBP)· [Total Vitamin D] + K_(alb) · [Alb] + 1 c = −[Total Vitamin D] 8[D_(free)] = [−b + √b2 − 4ac] ÷ 2a 9 [D_(bioavailable)] = [D_(free)] +[D_(Alb)] = (K_(alb) · [Alb] + 1) · [D_(free)] [Total Vitamin D] = totalmeasured vitamin D concentration (either 25-OH or 1.25-(OH)₂ vitamin D)[Alb] = measured albumin concentration [Total DBP] = measured vitamin Dbinding protein concentration [D_(Alb)] = concentration of albumin-boundvitamin D [D_(DBP)] = concentration of DBP-bound vitamin D [D_(free)] =concentration of free (unbound) D [D_(bioavailable)] = concentration ofBioavailable D = [D_(free)] + [D_(Alb)] K_(alb) = affinity constantbetween vitamin D and albumin = 6 × 10⁵M⁻¹ (for 25-OH D) or 5.4 × 10⁴M⁻¹(for 1.25-(OH)₂ D) K_(DBP) = affinity constant between vitamin D and DBF= 7 × 10⁸M⁻¹ (for 25-OH D) or 3.7 × 10⁷M⁻¹ (for 1.25-(OH)₂ D)

TABLE 9 Comparison of 25(OH)D insufficiency in blacks vs. whites basedon current definitions % 25(OH)D Deficient 25(OH)D Population Age(years) Blacks Whites Cutoff Levels Reference NHANES 2001-2006 >45(82-85%) 80 40 <20 ng/mL Diaz⁵¹ Older men (MrOS) >65 65 23 <20 ng/mLOrwoll¹⁷ 22 2 <10 ng/mL Hemodialysis 63 ± 15 31 12 <10 ng/mL Wolf⁴³patients (ArMORR) Men in BACH/Bone 30-79 44 11 <21 ng/mL Hannan¹⁹ SurveyNHANES = National Health and Nutrition Examination Survey; MrOS =Osteoporotic Fractures in Men Study; ArMORR = Accelerated Mortality onRenal Replacement; BACH = Boston Area Community Health

SEQUENCE LISTINGSEQ ID NO: 01 Human VDBP Isoform 1, variant 1 NCBI Ref: NP_000574   1mkrvlvllla vafghalerg rdyeknkvck efshlgkedf tslslvlysr kfpsgtfeqv  61sqlvkevvsl teaccaegad pdcydtrtsa lsakscesns pfpvhpgtae cctkeglerk 121lcmaalkhqp qefptyvept ndeiceafrk dpkeyanqfm weystnygqa plsllvsytk 181sylsmvgscc tsasptvcfl kerlqlkhls llttlsnrvc sqyaaygekk srlsnlikla 241qkvptadled vlplaeditn ilskccesas edcmakelpe htvklcdnls tknskfedcc 301qektamdvfv ctyfmpaaql pelpdvelpt nkdvcdpgnt kvmdkytfel srrthlpevf 361lskvleptlk slgeccdved sttcfnakgp llkkelssfi dkgqelcady sentfteykk 421klaerlkakl pdatptelak lvnkhsdfas nccsinsppl ycdseidael knilSEQ ID NO: 02 Human VDBP Isoform 1, variant 2 NCBI Ref: NP_001191235   1mkrvlvllla vafghalerg rdyeknkvck efshlgkedf tslslvlysr kfpsgtfeqv  61sqlvkevvsl teaccaegad pdcydtrtsa lsakscesns pfpvhpgtae cctkeglerk 121lcmaalkhqp qefptyvept ndeiceafrk dpkeyanqfm weystnygqa plsllvsytk 181sylsmvgscc tsasptvcfl kerlqlkhls llttlsnrvc sqyaaygekk srlsnlikla 241qkvptadled vlplaeditn ilskccesas edcmakelpe htvklcdnls tknskfedcc 301qektamdvfv ctyfmpaaql pelpdvelpt nkdvcdpgnt kvmdkytfel srrthlpevf 361lskvleptlk slgeccdved sttcfnakgp llkkelssfi dkgqelcady sentfteykk 421klaerlkakl pdatptelak lvnkhsdfas nccsinsppl ycdseidael knilSEQ ID NO: 03 Human VDBP Isoform 2 NCBI Ref: NP_001191236   1mlwswseerg gaarlsgrkm krvlvlllav afghalergr dyeknkvcke fshlgkedft  61slslvlysrk fpsgtfeqvs qlvkevvslt eaccaegadp dcydtrtsal sakscesnsp 121fpvhpgtaec ctkeglerkl cmaalkhqpq efptyveptn deiceafrkd pkeyanqfmw 181eystnygqap lsllvsytks ylsmvgscct sasptvcflk erlqlkhlsl lttlsnrvcs 241qyaaygekks rlsnliklaq kvptadledv lplaeditni lskccesase dcmakelpeh 301tvklcdnlst knskfedccq ektamdvfvc tyfmpaaqlp elpdvelptn kdvcdpgntk 361vmdkytfels rrthlpevfl skvleptlks lgeccdveds ttcfnakgpl lkkelssfid 421kgqelcadys entfteykkk laerlkaklp datptelakl vnkhsdfasn ccsinspply 481cdseidaelk nil SEQ ID NO: 04 Human preproalbumin NCBI Ref: NP_000468   1mkwvtfisll flfssaysrg vfrrdahkse vahrfkdlge enfkalvlia faqylqqcpf  61edhvklvnev tefaktcvad esaencdksl htlfgdklct vatlretyge madccakqep 121ernecflqhk ddnpnlprlv rpevdvmcta fhdneetflk kylyeiarrh pyfyapellf 181fakrykaaft eccqaadkaa cllpkldelr degkassakq rlkcaslqkf gerafkawav 241arlsqrfpka efaevsklvt dltkvhtecc hgdllecadd radlakyice nqdsissklk 301eccekpllek shciaevend empadlpsla adfveskdvc knyaeakdvf lgmflyeyar 361rhpdysvvll lrlaktyett lekccaaadp hecyakvfde fkplveepqn likqncelfe 421qlgeykfqna llvrytkkvp qvstptivev srnlgkvgsk cckhpeakrm pcaedylsvv 481lnqlcvlhek tpvsdrvtkc cteslvnrrp cfsalevdet yvpkefnaet ftfhadictl 541sekerqikkq talvelvkhk pkatkeqlka vmddfaafve kcckaddket cfaeegkklv 601aasqaalgl SEQ ID NO: 05 Human proalbuminrg vfrrdahkse vahrfkdlge enfkalvlia faqylqqcpfedhvklvnev tefaktcvad esaencdksl htlfgdklct vatlretyge madccakqepernecflqhk ddnpnlprlv rpevdvmcta fhdneetflk kylyeiarrh pyfyapellffakrykaaft eccqaadkaa cllpkldelr degkassakq rlkcaslqkf gerafkawavarlsqrfpka efaevsklvt dltkvhtecc hgdllecadd radlakyice nqdsissklkeccekpllek shciaevend empadlpsla adfveskdvc knyaeakdvf lgmflyeyarrhpdysvvll lrlaktyett lekccaaadp hecyakvfde fkplveepqn likqncelfeqlgeykfqna llvrytkkvp qvstptlvev srnlgkvgsk cckhpeakrm pcaedylsvvlnqlcvlhek tpvsdrvtkc cteslvnrrp cfsalevdet yvpkefnaet ftfhadictlsekerqikkq talvelvkhk pkatkeqlka vmddfaafve kcckaddket cfaeegkklvaasqaalgl SEQ ID NO: 06 Human albumindahkse vahrfkdlge enfkalvlia faqylqqcpfedhvklvnev tefaktcvad esaencdksl htlfgdklct vatlretyge madccakqepernecflqhk ddnpnlprlv rpevdvmcta fhdneetflk kylyeiarrh pyfyapellffakrykaaft eccqaadkaa cllpkldelr degkassakq rlkcaslqkf gerafkawavarlsqrfpka efaevsklvt dltkvhtecc hgdllecadd radlakyice nqdsissklkeccekpllek shciaevend empadlpsla adfveskdvc knyaeakdvf lgmflyeyarrhpdysvvll lrlaktyett lekccaaadp hecyakvfde fkplveepqn likqncelfeqlgeykfqna llvrytkkvp qvstptivev srnlgkvgsk cckhpeakrm pcaedylsvvlnqlcvlhek tpvsdrvtkc cteslvnrrp cfsalevdet yvpkefnaet ftfhadictlsekerqikkg talvelvkhk pkatkeqlka vmddfaafve kcckaddket cfaeegkklvaasqaalgl

What is claimed herein:
 1. A method for determining a level ofbioavailable vitamin D and a level of free vitamin D in a subject, themethod comprising: performing assays on a blood sample obtained from thesubject to determine a level of VDBP (vitamin D binding protein)polypeptide, albumin polypeptide and total vitamin D; calculating alevel of bioavailable vitamin D, wherein a level of bioavailable vitaminD is:=(Kalb*[Alb]+1)*[Free Vitamin D] and calculating a level of free vitaminD, wherein a level of free vitamin D is:={−{K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1}+√{(K _(DBP)·[Total DBP]−K _(DBP)·[Total Vitamin D]+K _(alb)·[Alb]+1)²+4·(K _(DBP) ·K _(alb) ·[Alb]+K _(DBP))·([Total VitaminD])}}÷(2·{K _(DBP) ·K _(alb) ·[Alb]+K _(DBP)}).
 2. The method of claim1, wherein a level of bioavailable vitamin D lower than 25% of the meanvalue of bioavailable vitamin D in a population of healthy subjectsindicates that the subject has a vitamin D insufficiency, and the methodincludes administering a vitamin D insufficiency treatment to a subjectwho is determined to have a vitamin D insufficiency.
 3. The method ofclaim 1, wherein the vitamin D is selected from the group consisting of:25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.
 4. The method of claim1, wherein the assays to determine the level of VDBP polypeptide oralbumin polypeptide comprises use of a method selected from the groupconsisting of: enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; dye linked immunosorbent assay;immunoturbidimetric assay; immunonephelometric assay; dye-basedphotometric assay; western blot; immunoprecipitation; radioimmunologicalassay (RIA); radioimmunometric assay; immunofluorescence assay and massspectroscopy.
 5. The method of claim 1, wherein the assays to determinethe level of total vitamin D comprises the use of a method selected fromthe group consisting of: radioimmunoassay; liquid chromatography tandemmass spectroscopy; enzyme linked immunosorbent assay; chemiluminescentimmunosorbent assay; electrochemiluminescent immunosorbent assay;fluorescent immunosorbent assay; and high-pressure liquidchromatography.
 6. The method of claim 2, wherein an insufficiency ofvitamin D indicates an increased risk of a condition selected from thegroup consisting of: decreased bone density; decreased bone mineraldensity; bone fractures; bone resorption; rickets; osteitis fibrosacystica; fibrogenesis imperfect ossium; osteoschlerosis; osteoporosis;osteomalacia; elevated parathyroid hormone levels; parathyroid glandhyperplasia; secondary hyperparathyroidism; hypocalcemia; infection;cancer; psoriasis; cardiovascular disease; renal osteodystrophy; renaldisease; end-stage renal disease; chronic kidney disease; chronic kidneydisease-associated mineral and bone disorder; extraskeletalcalcification; obesity; allergy, asthma; multiple sclerosis; muscleweakness; rheumatoid arthritis and diabetes.
 7. The method of claim 2,wherein the treatment comprises administering a compound selected fromthe group consisting of: calcitriol; dihydrotachysterol;doxercalciferol; paricalcitol; cholecalciferol and ergocalciferol. 8.The method of claim 1, wherein level of bioavailable vitamin D and thelevel of free vitamin D are calculated by a processor.